Amersham CyDye DIGE Fluor Labeling Kit for Scarce Samples

Amersham CyDye DIGE Fluor Labeling Kit for Scarce Samples
GE Healthcare
Amersham
CyDye DIGE Fluor Labeling
Kit for Scarce Samples
Reagents for labeling protein with CyDye DIGE Fluor Cy3 and Cy5
saturation dyes, before 2-dimensional electrophoresis
Product Booklet
Codes:
25-8009-83
25-8009-84
28-9366-83
Page finder
1. Legal
4
2.
6
6
6
6
Handling 2.1. Safety warnings and precautions 2.2. Storage
2.3. Expiry
3. Components
7
4. Other materials required
8
5.
Introduction
5.1. Description
5.2. Dye characteristics
5.3. Labeling with CyDye DIGE Fluor saturation dyes
5.4. Spot picking
5.5. Protein identification
5.6. Ettan DIGE system workflow
5.7. Measurement of variation
5.8. Experimental design
6.
Protocol
24
6.1. Introduction
26
6.2. Preparation of a cell lysate compatible with
saturation labeling
26
6.3. Determining the optimum amount of TCEP/dye
required to label a protein lysate
29
6.4. Reconstitution of CyDye DIGE Fluor saturation dyes in dimethylformamide (DMF)
36
6.5. Saturation labeling of a protein sample
37
6.6. First dimension isoelectric focusing of labeled
proteins
42
6.7. Second dimension SDS-PAGE electrophoresis
47
6.8. Preparing Ettan DALT preparative gels
50
6.9. Scanning CyDye DIGE Fluor saturation dye gels
using Typhoon Variable Mode Imager
51
2
11
11
13
14
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17
18
19
6.10. Image analysis using DeCyder 2-D Differential
Analysis Software
7.
Additional information
7.1. Requirements for cell lysis buffer
7.2. Protein lysate sonication
7.3. Adjustment of protein sample pH
7.4. Cell and tissue types tested with CyDye DIGE Fluor
saturation labeling
7.5. Reagents tested for compatibility with CyDye DIGE
Fluor saturation labeling
8. Troubleshooting guide 52
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63
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9. References 71
10.Related products 72
3
1. Legal
GE, imagination at work and GE monogram are trademarks of
General Electric Company.
Amersham, Ettan, Cy, CyDye, DeCyder, ImageQuant, Immobiline,
IPGphor, Multiphor, Pharmalyte, PlusOne, and Typhoon are
trademarks of GE Healthcare companies.
All third party trademarks are the property of their respective
owners.
2-D Fluorescence Difference Gel Electrophoresis:
2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE)
technology is covered by US patent numbers 6,043,025, 6,127,134
and 6,426,190 and equivalent patents and patent applications
in other countries and exclusively licensed from Carnegie Mellon
University.
CyDye:
This product or portions thereof is manufactured under an exclusive
license from Carnegie Mellon University under US patent numbers
5,569,587, 5,627,027 and equivalent patents in other countries. The
purchase of CyDye DIGE Fluors includes a limited license to use the
CyDye DIGE Fluors for internal research and development, but not
for any commercial purposes. A license to use the CyDye DIGE Fluors
for commercial purposes is subject to a separate license agreement
with GE Healthcare.
DeCyder:
This release of DeCyder version 7 (software) is provided by GE
Healthcare to the customer under a non-exclusive license and is
subject to terms and conditions set out in the 2-D Differential Gel
Electrophoresis Technology Access Agreement. Customer has no
rights to copy or duplicate or amend the Software without the prior
written approval of GE Healthcare.
© 2003–2006 General Electric Company – All rights reserved.
Previously published 2003
All goods and services are sold subject to the terms and conditions
of sale of the company within GE Healthcare which supplies them. A
copy of these terms and conditions is available on request.
Contact your local GE Healthcare representative for the most current
information.
www.gehealthcare.com/lifesciences
GE Healthcare UK Limited
Amersham Place, Little Chalfont,
Buckinghamshire, HP7 9NA UK
2. Handling
2.1. Safety warnings
and precautions
CAUTION: These dyes are
intensely colored and very
reactive. Care should be
exercised when handling the
dyes to avoid staining clothing,
skin, and other items. The
toxicity of CyDye™ DIGE Fluor
Cy™3 and Cy5 saturation dyes
has not yet been evaluated.
Warning: For research use
only. Not recommended
or intended for diagnosis
of disease in humans or
animals. Do not use internally
or externally in humans or
animals.
2.2. Storage
All chemicals should be
considered as potentially
hazardous. We therefore
recommend that this product is
handled only by those persons
who have been trained in
laboratory techniques and
that it is used in accordance
with the principles of good
laboratory practice. Wear
suitable protective clothing
such as laboratory overalls,
safety glasses and gloves.
Care should be taken to avoid
contact with skin or eyes. In
the case of contact with skin
or eyes wash immediately
with water. See material safety
data sheet(s) and/or safety
statement(s) for specific advice.
Store at -15°C to -30°C.
Avoid exposure to light, store in
the dark.
2.3. Expiry
For expiry date see outer
packaging.
Note: After reconstitution,
CyDye DIGE Fluor saturation
dyes are only stable and usable
until the expiry date detailed
on the tube or for 8 weeks,
whichever is sooner.
6
3. Components
25-8009-83: CyDye DIGE Fluor Labeling Kit for Scarce Samples
containing:
• 100 nmol CyDye DIGE Fluor Cy3 saturation dye for analytical
labeling;
• 100 nmol CyDye DIGE Fluor Cy5 saturation dye for analytical
labeling.
25-8009-84: CyDye DIGE Fluor Labeling Kit for Scarce Samples plus
Preparative Gel Labeling containing:
• 100 nmol CyDye DIGE Fluor Cy3 saturation dye for analytical
labeling;
• 100 nmol CyDye DIGE Fluor Cy5 saturation dye for analytical
labeling;
• 400 nmol CyDye DIGE Fluor Cy3 saturation dye for preparative
labeling.
28-9366-83: CyDye DIGE Fluor Preparative Gel Labeling containing:
• 400 nmol CyDye DIGE Fluor Cy3 saturation dye for preparative
labeling.
7
4. Other materials required
• Standard cell wash
buffer:
10 mM Tris (pH 8.0), 5 mM magnesium
acetate. Store at 2–8°C. Stable for 1
month.
• Cell lysis buffer:
30 mM Tris, 7 M urea, 2 M thiourea,
4% (w/v) CHAPS. Adjust to pH 8.0 with
1.0 M HCl. Aliquot and store at -15°C to
-30°C. Stable for 3 months
• pH indicator strips :
(Sigma‘ pH test strips pH 4.5-10.0 P-4536)
• 50 mM sodium
hydroxide (NaOH)
• Detergent compatible
reagent for protein
quantification:
We recommend Protein Determination
Reagent (USB, code 30098)
Reconstitution of dye and protein labeling
• 99.8% anhydrous
dimethylformamide
(DMF)
Must be less than 3 months old from day
of opening (Aldrich 22,705-6)
• Tris-(2-carboxyethyl)
phosphine
hydrochloride (TCEP)
(Molecular Probes, T-2556)
• 1 × sample buffer
(DTT/Pharmalytefree):
7 M Urea, 2 M Thiourea, 4% (w/v) CHAPS.
Dispense in aliquots and store at -15°C
to -30°C. Stable for 6 months.
• 2 × sample buffer:
7 M urea, 2 M thiourea, 4% (w/v) CHAPS,
2% (v/v) Pharmalyte™, broad range
pH 3-10, 130 mM DTT. Prepare fresh by
adding DTT and Pharmalytes to
8
1 × sample buffer. Use immediately and
discard any unused material.
Isoelectric focusing
• Rehydration buffer:
7 M urea, 2 M thiourea, 4% (w/v) CHAPS,
1% (v/v) Pharmalyte, broad range
pH 3-10, 13 mM DTT. Prepare fresh by
adding DTT and Pharmalytes to
1 × sample buffer. Use immediately and
discard any unused material.
SDS-PAGE separation
• Equilibration buffer:
6 M urea, 0.1 M Tris, pH 8.0, 30% (v/v)
glycerol, 2% (w/v) SDS, 0.5% (w/v) DTT.
A DTT-free stock can be prepared
and is stable at room temperature
for 6 months. DTT should be added
immediately prior to use and any
unused material discarded.
• 12.5% acrylamide gel
(for Ettan DALT):
281 ml acrylamide/bis 40% (v/v), 225 ml
Tris (1.5 M pH 8.8), 9 ml 10% (w/v) SDS,
9 ml 10% (w/v) ammonium persulfate
(freshly prepared on day of use), 1.24
ml 10% (v/v) TEMED. Make up to 900 ml
with distilled water. 900 ml is sufficient
solution to prepare a complete set of 14
Ettan DALT gels.
• Displacement
solution:
375 mM Tris (pH 8.8), 50% (v/v) glycerol,
bromophenol blue (2 mg/100 ml).
Prepare fresh and use immediately. Do
not store.
9
• Agarose overlay
solution:
0.5% LMP agarose prep, 0.1%
(w/v) bromophenol blue in 1 × SDS
electrophoresis running buffer (see
opposite). Stable for 1 month at room
temperature.
• Water saturated
butanol:
Add 50 ml water to 50 ml butan-2-ol
until two layers are visible. Stable for 6
months at room temperature.
• 1 × SDS
electrophoresis
running buffer:
25 mM Tris, 192 mM Glycine, 0.2% (w/v)
SDS. Store at room temperature.
• Suitable
electrophoresis
system:
SE 600 Ruby gel system, Ettan
DALTtwelve gel system, Ettan DALTsix
gel system or equivalent electrophoresis
system.
• Bind Silane solution:
100 µl PlusOne Bind-Silane (code
17-1330-01) added to 80 ml ethanol,
2 ml glacial acetic acid and 18 ml water.
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5. Introduction
5.1. Description
2–Dimensional Fluorescence Difference Gel Electrophoresis (2–D
DIGE) is a method for pre-labeling protein samples prior to 2–D
electrophoresis for difference analysis (1) that enables multiplexing
within the same 2–D gel.
The protocol described here is designed to work using CyDye
DIGE Fluor saturation dyes that have been developed specifically
for use with scarce protein samples. CyDye DIGE Fluor saturation
dyes enable a full 2–D analysis of samples which under normal
circumstances may be more challenging due to limiting sample
quantities.
The technology is based upon the specific properties of CyDye DIGE
Fluor Cy3 and Cy5 saturation dyes. The dyes are spectrally resolvable
so they can be detected independently using dye-specific imaging
parameters. CyDye DIGE Fluor Cy3 and Cy5 saturation dyes are
also migration-matched so identical proteins labeled with each of
the two CyDye DIGE Fluor saturation dyes will migrate to the same
position on a 2–D gel. These combined properties allow two different
protein samples to be labeled, one with each dye, separated on
the same gel and co-detected. The ability to multiplex permits the
inclusion of both sample and internal standard (internal reference)
in every gel. The use of an internal standard within each gel, helps
to limit system variation, which ultimately provides more accurate
quantitation of relative protein abundance.
Ettan DIGE system has capitalized on the ability to multiplex by
combining CyDye DIGE Fluor saturation dyes with DeCyder 2-D
Differential Analysis Software. This software has been designed
specifically for 2–D DIGE applications and utilizes a proprietary
co-detection algorithm that permits automatic detection,
11
background subtraction, quantitation, normalization, and inter-gel
matching of multiplexed fluorescent images. The major benefit
of this approach is the ability to produce quantitative data of
unparalleled accuracy, supported by statistical analysis. The use
of an in-gel standard increases confidence that the results reflect
true induced biological changes (i.e. due to a disease state or drug
treatment) and are not due to system variation.
Figure 1. Outline of the basic Ettan DIGE system for saturation
labeling
The system comprises CyDye DIGE Fluor saturation dyes for protein
labeling; a choice of Ettan IPGphor Isoelectric Focusing System or
Multiphor™ II IEF System for first-dimension separation; SE 600 Ruby,
Ettan DALTtwelve, or Ettan DALTsix vertical electrophoresis systems
for second-dimension separation; Typhoon 9000 series Variable
Mode Imager for advanced imaging; and DeCyder 2-D Differential
Analysis Software for quantitation and statistical analysis of protein
differences.
More detailed information and protocols for working with Ettan DIGE
12
system can be found in the Ettan DIGE System User Manual (code
18-1173-17). This manual is also available on the GE Healthcare
website (www.ettandige.com).
5.2. Dye characteristics
CyDye DIGE Fluor Cy3 saturation dye Molecular formula
C37H44N4O6S
Formula weight
672.85
Absorption max (in DMF)
548 ± 3 nm
Emission max (in DMF)
560 ± 5 nm
Structure confirmed by NMR
Figure 2. Absorption and emission spectra for CyDye DIGE Fluor Cy3
saturation dye in DMF
CyDye DIGE Fluor Cy5 saturation dye Molecular formula
C38H44N4O6S
Formula weight
684.86
Absorption max (in DMF)
641 ± 3 nm
Emission max (in DMF)
660 ± 5 nm
Structure confirmed by NMR
13
Figure 3. Absorption and emission spectra for CyDye DIGE Fluor Cy5
saturation dye in DMF
5.3. Labeling with CyDye DIGE Fluor
saturation dyes
Two dyes are available for saturation labeling, CyDye DIGE Fluor,
Cy3 and Cy5 saturation dyes. CyDye DIGE Fluor saturation dyes
have a maleimide reactive group which is designed to form a
covalent bond with the thiol group of cysteine residues on proteins
via a thioether linkage. To achieve maximal labeling of cysteine
residues, a high dye-to-protein labeling ratio is required. This type of
labeling method aims to label all available cysteines on each protein
under the conditions used, resulting in the majority of protein in a
sample being labeled. For this reason, this method has been called
“saturation” labeling.
The dyes offer great sensitivity with detection over 5 orders of
magnitude. Narrow excitation and emission bands mean that the
dyes are spectrally distinct, which makes them ideal for multicolor
detection. Most importantly, the dyes are migration-matched so
that the same protein labeled with either of the CyDye DIGE Fluor
14
saturation dyes will migrate to the same position within a 2–D gel.
The novel properties of the CyDye DIGE Fluor saturation dyes make
them ideal for multiplexing different protein samples within the same
2–D gel. This permits inclusion of an internal standard within each
gel which limits experimental variation and ensures accurate intergel matching.
Protein
1. TCEP
37°C, 1 hour, dark
Dye
Protein
2. 37°C, 30 minutes, dark
3. 2 × sample buffer
Dye
Protein
Figure 4. Schematic of labeling reaction between CyDye DIGE Fluor
saturation dye and the cysteine residue of a protein.
Many thiol groups on the cysteine residues in proteins exist as
disulphide bonds. In order to label these groups the protein must
be unfolded and the disulphide bonds broken. This can be achieved
15
under denaturing conditions with a reducing agent such as tris(2-carboxyethyl) phosphine hydrochloride (TCEP) and by increasing
the temperature of labeling. In some proteins, cysteine residues are
buried within the protein such that they cannot be reduced and are
not available for labeling. Thus the extent of labeling of cysteine
residues will depend on the accessibility of cysteines within the
protein under the reaction conditions used.
The cysteine amino acid in proteins has neutral charge at neutral or
acidic pH. CyDye DIGE Fluor saturation dyes are net neutral, ensuring
that the pI of the protein does not significantly alter on labeling. The
extent of the mass shift of a labeled protein depends on the cysteine
content of the protein and the accessibility of the cysteine residues
to dye in the labeling reaction.
5.4. Spot picking
Samples prepared using the saturation labeling approach can be
picked directly from a preparative gel. This eliminates the need for
post-staining.
5.5. Protein identification
Labeling of proteins with CyDye DIGE Fluor saturation dyes does
not affect identification by mass spectrometry. Labeling on cysteine
residues does not reduce the efficiency or specificity of enzymatic
digestion. Cysteine-labeled proteins generate equivalent levels of
peptide mass fingerprint (PMF) and sequence data to unlabeled
proteins.
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5.6. Ettan DIGE system workflow
The main steps in the Ettan DIGE system workflow for saturation
labeling are outlined below.
Figure 5. Ettan DIGE system workflow for saturation labeling
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5.7. Measurement of variation
2–D electrophoresis experiments experience variation that arises
from two main sources.
System variation may arise from two areas.
Firstly, gel-to-gel variation can result from differences in IEF and
electrophoretic running conditions between different gels, gel
distortions and user-to-user variation. The second source of system
variation is due to user-specific editing and interpretation when
using data analysis software.
Inherent biological variation arises from intrinsic differences that
occur within populations. For example, differences from animalto-animal, plant-to-plant or culture-to-culture which have been
subjected to identical conditions.
If induced biological changes, (the differences that are caused
by a disease state/drug treatment/life-cycle stage etc.) are to be
identified, it is important to be able to differentiate them from both
system variation and inherent biological variation.
System variation cannot be overcome when using conventional “one
sample per gel” 2-D electrophoresis. Ettan DIGE system controls
system variation by the inclusion of an internal standard within each
gel, enabled by the multiplexing capability of 2-D DIGE methodology.
Software-originated variation is minimized using DeCyder 2-D
Differential Analysis Software. This provides automated co-detection,
background subtraction, quantitation, normalization and inter-gel
matching, which limits user intervention and subjective editing,
generating consistent data.
To account for inherent biological variation, it is strongly advised
that biological replicates, such as multiple cultures, should be
incorporated into the experimental design. The more biological
replicates included in the experiment, the greater the chance that
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inherent biological variation will be taken into account, enabling a
reliable measure of induced biological change.
Since Ettan DIGE system variation is low by virtue of the inclusion of
an internal standard and the analysis method, biological variation
will far exceed the system variation. As a consequence, gel replicates
are no longer necessary.
5.8. Experimental design
To maximize the benefits of Ettan DIGE system, an internal standard
should be incorporated within each gel. The ideal internal standard
should comprise an aliquot from each biological sample within
the experiment. Thus, the internal standard is a pooled sample
created from all of the experimental samples. The internal standard
is labeled with one CyDye DIGE Fluor saturation dye (Cy3) and is
run on every gel together with experimental samples labeled with
the other CyDye DIGE Fluor saturation dye (Cy5) (see Table 1). This
ensures that every spot on every gel is represented within the
common internal standard. Using DeCyder 2-D Differential Analysis
Software each protein spot in a sample can be compared to its
representative within the internal standard to generate a ratio of
relative abundance (Figure 6).
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Figure 6. Quantitation of protein abundance using co-detection
algorithms in DeCyder 2-D Differential Analysis Software. From
each gel, two scan images are generated, Cy3 (red) for the internal
standard, and Cy5 (blue) for test samples. The protein abundance of
each spot in each sample is expressed as a normalized ratio relative
to spots from the in-gel internal standard
The same internal standard is run on all gels within the experimental
series. Matching of these internal standards creates an intrinsic
link between the samples on each of the different gels (Figure 7).
Quantitative comparisons of samples between gels are made based
on the relative change of sample to its in-gel internal standard. This
removes inter-gel (system) variation, a common problem associated
with traditional 2–D electrophoresis studies, enabling accurate,
statistical quantitation of induced biological change between
samples. For 2-D electrophoresis, Ettan DIGE system is the only
protein difference analysis technique that utilizes this approach (2).
20
Figure 7. Matching and comparison of samples across gels. In
DeCyder Differential Analysis Software the internal standard sample,
present on every gel, is used to aid matching of spot patterns
across all gels. The relative ratios of individual sample spots to their
internal standards is used to accurately compare protein abundance
between samples on different gels.
Linking every sample in-gel to a common standard offers many
advantages:
• accurate quantitation and spot statistics for changes in protein
abundance;
• increased confidence in matching between gels;
• flexibility of statistical analysis depending on the relationship
between samples;
• separation of induced biological variation from system variation.
For a more detailed guide to the benefits of using an internal
standard, see the Ettan DIGE System User Manual (code 18-1173-17.
Also available on the GE Healthcare website, www.ettandige.com).
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Table 1 shows an example of a recommended experimental set-up
designed to derive statistical data on differences between control
and two treatment regimens A and B. For the control and two
treatment regimens, three biological replicates are included (1–3).
The internal standard (a pool of equal amounts from all samples:
three control and six treated) is labeled with CyDye DIGE Fluor Cy3
saturation dye and run on every gel. Care should be taken to ensure
that there is sufficient sample to allow for preparation of the internal
standard.
Each control and treated test sample is labeled with CyDye DIGE
Fluor Cy5 saturation dye and loaded on gels as indicated below.
Gel
Cy3
Cy5
1
Pooled standard
Control 1
2
Pooled standard
Control 2
3
Pooled standard
Control 3
4
Pooled standard
Sample A1
5
Pooled standard
Sample A2
6
Pooled standard
Sample A3
7
Pooled standard
Sample B1
8
Pooled standard
Sample B2
9
Pooled standard
Sample B3
Table 1. Recommended experimental design for a 2–D DIGE saturation
labeling experiment, incorporating an internal standard. Each gel
contains a CyDye DIGE Fluor Cy3 saturation labeled standard which is
a pool of aliquots taken from each sample. Three biological replicates
(1–3) have been included for control and treated (A and B) samples
which are each labeled with a CyDye DIGE Fluor Cy5 saturation dye. For
further information relating to experimental design, please refer to the
Ettan DIGE System User Manual (code 18-1173-17).
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It is strongly advised that biological replicates are included in every
experimental group. This will enable accurate measurement of
the change due to a treatment/disease that is significant above
a baseline of inherent biological variation. The more biological
replicates, the more inherent biological variation is accounted for
and therefore, the more meaningful the results. Without biological
replicates, results are not biologically relevant and it is only possible
to conclude that differences are above system variation. Ettan DIGE
system variation is so low due to the internal standard and method
of analysis, that gel replicates are not needed - any system variation
should be far outweighed by the inherent biological variation. Gel
replicates can be included if the user wishes.
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6. Protocol
For users familiar with CyDye DIGE Fluor minimal labeling, please
note the following key differences between minimal labeling and
saturation labeling experiments before starting.
Saturation labeling
Minimal labeling
Sample
Cell lysis buffer is at
preparation pH 8.0.
(For a complete recipe see
page 8, “Other materials
required”).
Cell lysis buffer is at
pH 8.5.
Dyes
Maleimide dyes.
Label cysteine residues.
2 dyes available.
NHS ester dyes.
Label lysine residues.
3 dyes available.
CyDye DIGE Fluor
saturation dyes are
reconstituted at 2 mM
(analytical gels) or 20 mM
(preparative gels) Once
reconstituted, the dyes are
stable for up to 8 weeks at
-15°C to -30°C.
Once reconstituted, the
concentrated stock (1 mM)
of CyDye DIGE Fluor
minimal dyes is stable for
up to 2 months at -15°C to
-30°C.
Once reconstituted, dyes
do not need to be diluted
further.
The working concentration
of the dyes is 0.4 mM and
is stable for 1 week.
Proteins must be reduced
using TCEP prior to
labeling.
No reduction step required.
Protein
labeling
24
Protein
labeling
Saturation labeling
Minimal labeling
Labeling reaction
performed at 37°C.
Labeling reaction
performed at 4°C.
Labeling reaction
quenched using 2 ×
sample buffer.
Labeling reaction
quenched with 10 mM
lysine.
Labeling is optimized by
titrating TCEP and dye
(Cy3 and Cy5) then
analyzing on a 2–D gel.
Labeling is optimized by
comparing labeled
samples on a 1-D gel.
Labeled proteins are
stable for 1 month at
-70°C.
Labeled proteins have
stability equivalent to
unlabeled protein at -70°C.
Protein
No iodoacetamide
separation equilibration step prior to
and analysis 2–DE.
A Cy3 labeled sample
is used to prepare a
preparative gel for spot
picking. No staining is
required.
Iodoacetamide
equilibration step required.
An unlabeled sample is
used to prepare a
preparative gel for spot
picking. The gel must be
stained using a fluorescent
post-stain to allow
matching to analytical gels
for picking.
Table 2. Key differences between minimal labeling and saturation
labeling experiments
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6.1. Introduction
This protocol provides all the information required for the use
of CyDye DIGE Fluor saturation dyes to label proteins prior to
2–D electrophoresis. It is recommended that the protocol is read
thoroughly before using the system and that it is followed precisely.
For recommended materials and recipes required for saturation
labeling and 2–D electrophoresis, please refer to page 8, “Other
materials required”.
In the standard labeling protocol, proteins are first solubilized in a
cell lysis buffer. The protein concentration should then be determined
using a standard protein quantitation method. Cysteine residues in
the extracted proteins are reduced by incubating with TCEP, at 37°C
for 1 hour. CyDye DIGE Fluor saturation dye is added to the protein
lysate and the reaction incubated at 37°C in the dark for a further
30 minutes. Finally, the reaction is quenched by the addition of 2 ×
sample buffer.
Plastic tubes should be used when handling samples, as many
proteins will adhere to glassware. The fluorescent properties of
CyDye DIGE Fluor Cy3 and Cy5 saturation dyes can be adversely
affected by exposure to light, so it is recommended that the
exposure of dye or labeled protein to all light sources is kept to a
minimum.
6.2. Preparation of a cell lysate compatible
with saturation labeling
Samples should be lysed in the recommended cell lysis buffer
(refer to page 8, “Other materials required”). The new 2-D Protein
Extraction Buffers are compatible with CyDye DIGE Fluors with the
following exception:
Buffer-III and -IV are not suitable when CyDye DIGE Fluor labeling
kit for scarce samples is used since the labeling efficiency is
26
significantly reduced. Care should be taken to exclude compounds
that may interfere with labeling. These include primary amines (e.g.
Pharmalytes or ampholytes) or thiols (e.g. DTT) which will compete
with the protein for dye.
The concentration of the lysate before labeling should be between
0.55–10 mg/ml for running analytical gels or 1.2–10 mg/ml for
running preparative gels (concentrations >1.2 mg/ml may be
required for lysates using high levels of TCEP and dye).
After lysis, the pH of the sample should be measured to check that
it has not deviated from pH 8.0 (refer to page 60, “Adjustment of
protein sample pH”).
Extraction protocol suitable for most tissue samples.
The extraction method given below is a general method suitable for
most tissue samples. Recommendations for extraction of different
cell and tissue types are listed on pages 60–64.
1. Wash the tissue in 0.9% saline solution
2. Add a small volume of cell lysis buffer (30 mM Tris, 7 M Urea, 2 M
Thiourea, 4% (w/v) CHAPS, pH 8.0).
Note: if the protein concentration is less than 0.55 mg/ml (for
analytical samples) or 1.2 mg/ml (for preparative samples) after
protein quantitation, resuspend cells in a correspondingly smaller
volume of cell lysis buffer in subsequent experiments.
3. Mechanically homogenize the sample.
4. Keep the sample on ice and sonicate intermittently until the
sample is lysed. This may be performed using the sample held
in a small vessel within a water bath sonicator, for extraction of
samples for analytical gels. For larger amounts of sample (e.g.
for preparative gels) use a probe sonicator, see page 58, “Protein
lysate sonication”.
Note: the cell suspension must be kept cool at all times.
27
5. Pellet the tissue in a microcentrifuge at 12 000 × g for 10 minutes
at 4°C.
6. Transfer supernatant to a labeled tube. This is the cell lysate to be
used for dye labeling. Discard the pellet. Check that the pH of the
cell lysate is still at pH 8.0 by spotting 1 µl on a pH indicator strip.
If the pH of the cell lysate has fallen below pH 8.0 then the pH of
the lysate will need to be adjusted before labeling. See page 60,
“Adjustment of protein sample pH”.
Store cell lysates in aliquots at -70°C until protein concentration is to
be determined.
Note: for determination of protein concentration, a detergent
compatible assay is recommended. We recommend Protein
Determination Reagent (USB, code 30098).
Extraction protocol suitable for laser capture microdissected
(LCM) samples.
The extraction method given below was used to prepare a lasercapture micro-dissected mouse hippocampus lysate.
1. Capture the section(s) directly into a small volume of cell lysis
buffer (30 mM Tris, 7 M Urea, 2 M Thiourea, 4% (w/v) CHAPS, 5 mM
magnesium acetate, pH 8.0). The section may be still attached to
the mount.
Note: if the protein concentration is less than 0.55 mg/ml (for
analytical samples) or 1.2 mg/ml (for preparative samples) after
protein quantitation, resuspend cells in a correspondingly smaller
volume of cell lysis buffer in subsequent experiments.
2. Keep the sample on ice and sonicate intermittently until the
sample is lysed. This may be performed using the sample held
in a small vessel within a water bath sonicator, for extraction of
samples for analytical gels. For larger amounts of sample (e.g.
for preparative gels) use a probe sonicator, see page 58, “Protein
lysate sonication”.
28
Note: the cell suspension must be kept cool at all times.
3. Pellet the tissue in a microcentrifuge at 9 000 × g for 30 seconds
at 4°C.
4. Transfer supernatant to a labeled tube. This is the cell lysate to be
used for dye labeling. Discard the pellet. Check that the pH of the
cell lysate is still at pH 8.0 by spotting 1 µl on a pH indicator strip.
If the pH of the cell lysate has fallen below pH 8.0 then the pH of
the lysate will need to be adjusted before labeling. See page 60,
“Adjustment of protein sample pH”.
Store cell lysates in aliquots at -70°C until protein concentration is to
be determined.
Note: for determination of protein concentration, a detergent
compatible assay is recommended. We recommend Protein
Determination Reagent (USB, code 30098).
6.3. Determining the optimum amount of
TCEP/dye required to label a protein lysate
The amount of TCEP and CyDye DIGE Fluor saturation dye used in
the labeling reaction needs to be determined individually for each
protein sample type being analyzed or when a non-standard cell
lysis buffer is being used. A labeling optimization experiment should
always be performed when:
• a new sample type is being used;
• the cell lysis buffer contains a reagent which hasn’t been tested for
compatibility with CyDye DIGE Fluor saturation labeling (see pages
65–67);
• the cell lysis buffer contains a reagent which has been tested
for compatibility with CyDye DIGE Fluor saturation labeling, but
is being used in a range known to affect labeling efficiency or is
being used outside the recommended concentration range;
29
• the cell lysis buffer contains a combination of reagents that may or
may not have been tested for compatibility with CyDye DIGE Fluor
saturation labeling. The effect of different reagents on labeling
efficiency is additive and may lead to unexpectedly poor labeling
when one or more interfering reagents are used together.
The molar ratio of TCEP:dye should always be kept at 1:2 to ensure
efficient labeling. Samples with higher cysteine content will require
more TCEP to reduce the disulphide bonds and more dye to label
the thiol groups. Typically, 5 µg of protein lysate requires 2 nmol
TCEP and 4 nmol dye for the labeling reaction (assuming an average
cysteine content of 2%). Mammalian samples, with a higher
glutathione content (e.g. liver tissue) may require more TCEP (e.g.
3 nmol) for the reduction step and therefore require more dye (e.g.
6 nmol).
Figure 8. Scheme showing the workflow for labeling optimization.
This should be repeated for 6 different TCEP/dye concentrations (see
Table 3).
30
To determine the optimum amount of TCEP and dye required for the
protein extract being used, a simple titration should be performed
for each dye before proceeding with any analytical experiments
(Figure 8). The protocol described below should be followed using the
pooled protein extract.
To prepare a pooled protein extract, mix equal amounts of each
experimental sample together. This extract is labeled using different
amounts of TCEP and CyDye DIGE Fluor Cy3 or Cy5 saturation dye,
as shown in Table 3. Cy3 or Cy5 labeled samples for the same
TCEP:dye concentration should be run on the same gel so a total of
6 gels are required.
Note: The molar ratio of TCEP:dye should always be kept at 1:2 to
ensure efficient labeling.
Gel
1
2
3
4
5
6
2 mM TCEP (nmol)
TCEP (µl)
0.5
0.75
1
1.25
1.5
2
1
1.5
2
2.5
3
4
2 mM Dye (µl)
Dye (nmol)
1
1.5
2
2.5
3
4
2
3
4
5
6
8
Table 3. Recommended amounts of TCEP and dye required for
labeling optimization, prior to analytical experiments. Amounts
shown in this table are for optimizing the labeling of 5 µg protein.
Typical TCEP/dye quantities required for 5 µg of protein are
highlighted. The recommended times for reduction and labeling
reactions allow different volumes of TCEP/dye to be used without
any adverse effects on reduction/labeling kinetics.
For each gel, create a red/blue Cy3/Cy5 image overlay e.g. using
ImageQuant™ or Paint Shop Pro™ (a product of Jasc Software).
31
Compare gel overlays along the titration series to decide which gel
gives the best labeling results.
The criteria for optimal labeling conditions are:
• all spots overlaid;
• no significant mass trains or vertical streaks;
• no significant charge trains or horizontal streaks.
If the amount of TCEP/dye is too low available thiol groups on
some proteins will not be labeled. When the maleimide dye labels
a thiol group, the mass of the protein is increased but the charge
is unaffected. Thus, under-labeled samples will show MW trains
and/or streaking in the vertical direction (see Figure 9b). Differential
migration of Cy3 and Cy5 labeled spots for the same protein can
also occur when the amount of TCEP/dye is too low.
If the amount of TCEP/dye is too high non-specific labeling of the
amine groups on lysine residues can occur. When the maleimide dye
labels a lysine group, the mass of the protein is increased and the
charge is also reduced by 1. Thus, over-labeled samples will show pI
charge trains and/or streaking in the horizontal direction.
When separating by SDS-PAGE, the migration of proteins in the
range 20–30 kDa is particularly sensitive to the fine structure of the
dyes attached to labeled proteins. This effect may result in a small
number of spots (typically less than 1% of all spots on a gel) which,
although labeled to the same extent, do not overlay on the dualcolor image. These proteins should be identified during the labeling
optimization experiments and spots merged when using DeCyder
2-D Differential Analysis Software, prior to performing statistical
analysis on analytical gels.
Note: If the overlays are visualized using the DeCyder DIA software,
some proteins will exhibit differential detection between the two
dyes. It is possible that this may be due to Cy5 quenching effects
32
with some highly labeled proteins (3). Use of the recommended
experimental design incorporating an internal standard (see Table 1)
will compensate for this phenomenon.
The method of protocol optimization described on page 33, for the
determination of dye/TCEP levels can also be used to test the effect
(or optimize the concentration) of additional components the user
may wish to add to the cell lysis buffer.
Figures 9a-d show gel images taken from a labeling optimization
experiment using rat liver, spiked with glutathione. The gels show
characteristic changes in the spot pattern when progressing from
non-optimal to optimal labeling conditions.
• Gel (b) shows vertical streaking, a characteristic typical of
underlabeling.
• The proteins highlighted in boxes show multiple spots for lower
TCEP/dye levels, each moving to a single spot using the optimum
labeling conditions, gel (d). This phenomenon is characteristic of
proteins which are incompletely labeled at lower TCEP/dye levels
but become fully saturated when optimum conditions are reached.
• In this example, the optimal labeling conditions are 5 µg protein:
4 nmol TCEP:8 nmol dye.
This example illustrates that samples containing glutathione may
require higher levels of TCEP and dye for optimal labeling to be
achieved.
Once the optimal labeling conditions have been established for a
particular sample, only those conditions should be used for further
work. If the sample type or sample preparation method changes the
labeling conditions will require re-optimization.
33
Figure 9a. Control rat liver (no glutathione) labeled with 2 nmol TCEP
and 4 nmol dye. Protein spots that were markedly changed when
glutathione was added to this sample (see below) are boxed.
Figure 9b. Rat liver (spiked with 3 nmol glutathione) labeled with
2 nmol TCEP and 4 nmol dye. The image shows vertical streaking as
a consequence of underlabeling
34
Figure 9c. Rat liver (spiked with 3 nmol glutathione) labeled with
3 nmol TCEP and 6 nmol dye. This image shows a reduction in vertical
streaking and increase in mass of some underlabeled proteins, as labeling
becomes more optimal.
Figure 9d. Rat liver (spiked with 3 nmol glutathione) labeled with
4 nmol TCEP and 8 nmol dye. This image shows optimal labeling
comparable to the glutathione-free liver sample shown in Figure 9a.
35
6.4. Reconstitution of CyDye DIGE Fluor
saturation dyes in dimethylformamide (DMF)
Each vial of CyDye DIGE Fluor saturation dye powder must be
reconstituted in high quality anhydrous DMF (specification: ≤ 0.005%
H2O, ≥ 99.8% pure) open for less than 3 months. On reconstitution in
DMF the CyDye DIGE Fluor will give a deep color; Cy3-red, Cy5-blue.
The quality of the DMF used in all experiments is critical to ensure
that protein labeling is successful. The DMF must be anhydrous
and every effort should be taken to ensure it is not contaminated
with water. DMF after opening, over a period of time, will degrade
with amine compounds being produced. Amines will react with the
maleimide dye, reducing the concentration available for protein
labeling. If in doubt use an unopened batch of DMF for reconstituting
the dye.
For analytical labeling reactions, the working dye solution should
be at a concentration of 2 mM. The volume of reconstituted dye
added depends on the amount of dye required, as determined in the
labeling optimization experiments.
For preparative labeling reactions, the working dye solution should
be at a concentration of 20 mM. The volume of reconstituted dye
added depends on the amount of dye required, as determined in the
labeling optimization experiments.
1. Take a small volume of DMF from its original container and
dispense into a fresh microfuge tube.
2. Remove the CyDye DIGE Fluor saturation dye from the -15°C to
-30°C freezer and leave unopened for 5 minutes, to warm to room
temperature.
3. Once at ambient temperature, add the required volume of DMF to
each new vial of CyDye DIGE Fluor saturation dye.
For 5 µg analytical labeling reactions, reconstitute 100 nmol dye
36
in 50 µl DMF to give a 2 mM working dye solution.
F or preparative labeling reactions, reconstitute 400 nmol dye in 20
µl DMF to give a 20 mM working dye solution.
4. Replace the cap on the dye microfuge tube and vortex vigorously
for 30 seconds.
5. Centrifuge for 30 seconds at 12 000 × g in a microcentrifuge.
6. The dye can now be used. Check that the dye solution is an
intense color. During transport, the dye powder may spread
around the inside surface of the tube (including the lid). If the dye
is not an intense color, then pipette the solution around the tube
(and lid) to ensure complete resuspension of dye. Vortex and spin
down.
Note: Unused dye stock solution should be returned to the -15°C to
-30°C freezer as soon as possible and stored in the dark.
After reconstitution CyDye DIGE Fluor saturation dyes are stable
and usable until the expiry date detailed on the tube, or for 8 weeks,
whichever is sooner.
6.5. Saturation labeling of a protein sample
The minimum requirement for protein concentration in the lysate
is DIFFERENT for analytical and preparative labeling reactions.
Protein lysates used for analytical labeling reactions must contain
>0.55 mg/ml protein to ensure the final volume for cup-loading
does not exceed 100 µl. The recommended concentration of protein
lysates used for preparative labeling is >1.2 mg/ml, when using
24 cm Immobiline DryStrips. This ensures that the volume used to
rehydrate the strip does not exceed the maximum recommended
rehydration volume (Table 4). Smaller strips require lower rehydration
volumes and will therefore require a higher starting concentration
of protein lysate. Higher protein concentrations may be required for
preparative gels if large amounts of TCEP and dye are used.
37
Immobiline DryStrip length (cm)
Total volume per strip (ml)
7
11
13
18
24
125
200
250
350
450
Table 4. Rehydration volumes of Immobiline DryStrips
Check that the pH of the cell lysate is still at pH 8.0 by spotting 1 µl
onto a pH indicator strip. If the pH of the cell lysate has deviated
from pH 8.0 then the pH of the lysate will need to be adjusted before
labeling (refer to page 60, “Adjustment of protein sample pH”).
Prepare a pooled internal standard by mixing equal amounts of each
experimental protein sample together, ensuring that there is enough
of the resulting pooled standard sample to include on each gel
within the experiment.
The labeling protocol required will depend on whether samples
are being labeled for analytical or preparative gels. Both methods
are given below. If you wish to scale up analytical or preparative
reactions for bulk labelings, remember to increase the amount of
TCEP and dye accordingly. Do this by adjusting the volume added
(not the reagent concentration) to reflect the amount of protein,
maintaining the same ratio of protein:TCEP:dye.
Labeling samples for analytical gels
For analytical labeling reactions, the dye and TCEP should both
be used at a concentration of 2 mM. The volume added for each
component depends on the amount of TCEP and dye required, as
determined in the labeling optimization experiment.
1. Add a volume of protein lysate equivalent to 5 µg protein to a
sterile microfuge tube.
2. Make up the volume to 9 µl with cell lysis buffer.
38
3. Prepare 2 mM TCEP solution by dissolving 2.8 mg TCEP in 5 ml
water. TCEP solution is unstable and should be used immediately.
Discard any unused material.
4. Add the required volume of 2 mM TCEP appropriate for 5 µg
protein (as determined in the labeling optimization experiment,
see page 30). For example add 1 µl TCEP (2 nmol).
5. Mix vigorously by pipetting.
6. Spin down the sample in a microcentrifuge and incubate at 37°C
for 1 hour, in the dark.
7. Add the required volume of resuspended 2 mM CyDye DIGE Fluor
saturation dye appropriate for 5 µg protein (as determined in the
labeling optimization experiment, see page 30). For example add
2 µl dye (4 nmol).
Label the pooled protein extract with CyDye DIGE Fluor Cy3
saturation dye.
Label experimental protein extracts (e.g. control, treated) with CyDye
DIGE Fluor Cy5 saturation dye.
8. Mix vigorously by pipetting.
9. S
pin down the sample in a microcentrifuge and incubate at 37°C
for 30 minutes, in the dark.
10. Whilst the sample is incubating, prepare 2 × sample buffer by
adding Pharmalytes (2% final) and DTT (130 mM final) to 1 ×
sample buffer (7 M Urea, 2 M Thiourea, 4% CHAPS).
11. To stop the reaction, calculate the total volume of the labeling
reaction and add an equal volume of 2 × sample buffer.
12. Mix vigorously by pipetting.
13. Spin down the sample in a microcentrifuge.
14. Samples are ready for use and can be stored on ice or frozen for
up to one month, at -70°C, in the dark.
39
Note: Protein lysates are viscous so failure to mix thoroughly at
steps 5, 8 and 12 can cause non-uniform labeling. This can result in
poor spot overlays due to pI differences and mass shifts between
Cy3 and Cy5 labeled protein spots. Vortexing is not recommended as
mixing is not adequate using this technique.
Labeling samples for preparative gels
For preparative gels, it is recommended that 500 µg of the
pooled internal standard sample is labeled using CyDye DIGE
Fluor Cy3 saturation dye. For applications where specific material
is very scarce (e.g. sections of brain prepared by laser capture
microdissection), material from surrounding tissue can be used for
the preparative gel. This approach assumes that the protein profile
will be similar for both localized and surrounding tissues.
For analytical labeling reactions in a small volume, Pharmalytes
and DTT are added as part of the 2 × sample buffer. However, for
preparative labeling, a larger volume of protein is required and in-gel
rehydration sample loading must be used. The maximum volume
that can be loaded by in-gel rehydration is 450 µl (for 24 cm strips).
This means that 1 × sample buffer, Pharmalytes and DTT must be
added separately.
For preparative labeling reactions, TCEP is added in a constant
volume of 10 µl and the dye is added at a constant concentration
of 20 mM. The concentration of TCEP and volume of dye used
depend on the amount of TCEP and dye required, as determined
in the labeling optimization experiments and should be scaled up
accordingly.
1. Add a volume of protein lysate (preferably the pooled internal
standard for a preparative gel) appropriate for 500 µg protein to
a sterile microfuge tube. For example, add 250 µl for a 2 mg/ml
protein lysate. If the volume of lysate is below 250 µl, then make
up the volume to 250 µl with lysis buffer before labeling.
40
2. Prepare TCEP solution at the required concentration appropriate
for 500 µg protein (as determined in the labeling optimization
experiment, see page 30).
For example, dissolve 2.8 mg TCEP in 500 µl water to give a
20 mM solution. TCEP solution is unstable and should be used
immediately. Discard any unused material.
3. Add 10 µl of TCEP to the protein lysate. For example add 10 µl
TCEP at 20 mM (200 nmol).
4. Mix vigorously by pipetting.
5. Spin down the sample in a microcentrifuge and incubate at 37°C
for 1 hour, in the dark.
6. Add the required volume appropriate for 500 µg protein of 20 mM
CyDye DIGE Fluor Cy3 saturation dye (as determined in the
labeling optimization experiment, see page 30).
For example add 20 µl dye at 20 mM (400 nmol).
7. Mix vigorously by pipetting.
8. Spin down the sample in a microcentrifuge and incubate at 37°C
for 30 minutes, in the dark.
9. To stop the reaction, add a volume of 1 × sample buffer (DTT/
Pharmalyte-free) to take the total volume up to 445.5 µl. For
example if your unlabeled protein started at a concentration of
2 mg/ml, you will need to add (445.5 - 250 - 10 - 20) = 165.5 µl of
1 × sample buffer.
10. Mix vigorously by pipetting.
11. Add 4.5 µl Pharmalytes pH 3-10 for IEF. The total volume should
now be 450 µl.
12. Mix vigorously by pipetting.
13. Add 4.5 mg DTT (final DTT concentration of 130 mM).
14. Mix vigorously by pipetting.
41
15. Spin down the sample in a microcentrifuge.
16. Samples are ready for use and can be stored on ice or frozen for
up to one month, at -70°C, in the dark.
Note: Protein lysates are viscous so failure to mix thoroughly after
reagent addition can cause non-uniform labeling. This can result
in poor preparative/analytical gel matching due to pI differences
and mass shifts between labeled protein spots. Vortexing is not
recommended.
6.6. First dimension isoelectric focusing of
labeled proteins
We recommend that labeled protein samples are loaded onto strips
using the cup-loading approach for analytical gels (5 µg of protein
per strip), or in-gel rehydration for preparative gels (up to 500 µg
protein per strip). The methods below describe sample application
using both of these techniques followed by focusing using Ettan
IPGphor IEF system. For loadings above 5 µg, labeling reactions
should be scaled up, maintaining the ratio of protein:TCEP:dye
determined in the labeling optimization experiment.
For general information and protocols for the use of Ettan IPGphor
IEF system and Multiphor II IEF system in 2–D DIGE experiments,
please refer to the Ettan DIGE System User Manual (code 18-1173-17).
For more detailed information on Ettan IPGphor IEF system and
Multiphor II IEF system please refer to the accompanying User
Manuals (code nos. 80-6415-35 and 18-1103-43 respectively).
Immobiline DryStrip rehydration for cup loading (analytical gels)
1. Pipette the appropriate volume of rehydration buffer into each
of the required number of slots in an IPGbox Reswell Tray. The
volume should not exceed the maximum volume determined for
each Immobiline DryStrip size, shown in Table 5.
42
Immobiline DryStrip
length (cm)
Total volume per strip (µl)
7
11
13
18
24
125
200
250
350
450
Table 5. Rehydration volumes of Immobiline DryStrips
2. Deliver the buffer slowly along the slot. Remove any large
bubbles.
3. Remove the protective cover from the Immobiline DryStrip.
4. Position the Immobiline DryStrip with the gel side down and
lower the Immobiline DryStrip onto the buffer. To help coat the
entire Immobiline DryStrip and avoid air bubbles, gently lift and
lower the strip along the surface of the buffer.
5. Close the lid of the IPGbox and allow the Immobiline DryStrips
to rehydrate at room temperature. A minimum of 10 hours is
required for rehydration; overnight is recommended, up to a
maximum of 24 hours.
Sample application using the cup-loading approach (analytical
gels)
1. Place precut electrode papers on a clean dry surface such as a
glass plate and soak with distilled water. Remove excess water
by blotting with a paper towel, or filter paper.
Note: It is important that the electrode papers are damp and not
wet. Excess water may cause streaking.
2. Combine the required amount of each labeled protein extract
(e.g. 5 µg Cy3 pooled internal standard, 5 µg Cy5 experimental
sample).
43
3. Mix thoroughly by pipetting and leave on ice until use.
4. Place the IPGphor Manifold in the correct position on the Ettan
IPGphor platform.
5. With a pair of forceps carefully remove the Immobiline DryStrip
from the IPGbox taking care not to damage the gel.
6. Place the Immobiline DryStrip gel side up with the acidic end of
the strips oriented toward the anodic side of the instrument.
7. Place a pre-cut damp electrode paper (from step 1) onto the
acidic and basic ends of the gel.
8. Clip down the electrodes firmly onto the electrode papers.
Ensure that there is good contact between the electrode papers
and the metal of the electrode.
9. Clip a loading cup onto the end of the strip. It should
be positioned either at the acidic or basic end (see
recommendations for IEF conditions on page 48), in between the
two electrodes.
10. To check for a good seal fill the cup to the top with PlusOne
Immobiline DryStrip Cover Fluid. Observe the level of the fluid to
check if it is decreasing. If a leak is detected remove the PlusOne
Immobiline DryStrip Cover Fluid and reposition the sample cup.
11. Apply 108 ml of PlusOne Immobiline DryStrip Cover Fluid
allowing the oil to spread so it completely covers the Immobiline
DryStrips.
12. Up to 100 µl of protein sample can now be loaded into the
bottom of the sample cup.
13. Close the lid of the Ettan IPGphor instrument.
Your strips are now ready for isoelectric focusing.
Immobiline DryStrip preparation using the in-gel rehydration
approach (high protein loads, e.g. preparative gels)
44
1. Deliver 500 µg labeled protein in a volume of 450 µl (for a 24
cm Immobiline DryStrip), slowly down the centre of the slot in
the IPGbox Reswell Tray. The volume should not exceed the
maximum volume determined for each Immobiline DryStrip size,
shown in Table 5. Remove any large bubbles.
2. Remove the protective cover from the Immobiline DryStrip.
3. Position the Immobiline DryStrip with the gel side down and
lower the Immobiline DryStrip onto the buffer. To help coat the
entire Immobiline DryStrip and avoid air bubbles, gently lift and
lower the strip along the surface of the buffer.
4. Close the lid of the IPGbox and allow the Immobiline DryStrips
to rehydrate at room temperature. A minimum of 10 hours is
required for rehydration; overnight is recommended, up to a
maximum of 24 hours.
5. Place precut electrode papers on a clean dry surface such as a
glass plate and soak with distilled water. Remove excess water
by blotting with a paper towel, or filter paper.
Note: It is important that the electrode papers are damp and not
wet. Excess water may cause streaking.
6. Place the Manifold in the correct position on the Ettan IPGphor
platform.
7. With a pair of forceps carefully remove the Immobiline DryStrip
from the IPGbox, taking care not to damage the gel.
8. Place the Immobiline DryStrip gel side up with the acidic end of
the strips oriented toward the acidic side of the instrument.
9. Place a pre-cut damp electrode pad paper (from step 1) onto the
acidic and basic ends of the gel.
10. Clip down the electrodes firmly onto the electrode papers.
Ensure that there is good contact between the electrode papers
and the metal.
45
11. Apply 108 ml of PlusOne Immobiline DryStrip Cover Fluid
allowing the oil to spread so it completely covers the Immobiline
DryStrips.
12. Close the lid of the Ettan IPGphor instrument.
Your strips are now ready for isoelectric focusing.
Isoelectric focusing using the IPGphor IEF system
Focus the proteins overnight. A typical program used for analytical
protein loads with 24 cm pH 3–10 strips, is shown in Table 6.
Power
Ramp
Duration
1
2
3
4
5
6
300 V
600 V
1000 V
8000 V
8000 V
500 V
Step-and-hold
Gradient
Gradient
Gradient
Step-and-hold
Step-and-hold
3 hours
3 hours
3 hours
3 hours
4 hours
48 hours
50 µA per strip, 25°C
Table 6. Suggested focusing program for use with the Ettan IPGphor
IEF system.
Strips should be removed as soon as possible after step 5 is
completed. If they are left for more than 2 hours at 500 V, strips
should be ramped up to 8000 V over 30 minutes to refocus proteins
before strips are removed.
Cathodic cup-loading may give better results for acidic IPG
strips and DeStreak Rehydration Solution (code 17-6003-19) is
recommended for use with IPG strips containing basic regions.
Higher protein loads (e.g. for preparative gels) may require longer
focusing times. More detailed guidelines for first dimension
conditions and focusing parameters are available in the Ettan DIGE
System User Manual (code 18-1173-17).
46
If the Immobiline DryStrip is not run immediately on the second
dimension gel, it can be stored at -70°C in a sealed container
(e.g. equilibration tube or petri dish). The container has to be rigid
because a frozen Immobiline DryStrip is very brittle and can easily
be damaged. Do not equilibrate Immobiline DryStrip prior to storage,
this must be carried out immediately before the second dimension
separation.
6.7. Second dimension SDS-PAGE electrophoresis
Important:
Low fluorescence glass plates must be used for 2–D DIGE
fluorescent gels. See related products list, page 74 for recommended
plates or precast DIGE gels.
To prepare a gel for spot picking, attach reference markers to the
glass plate treated with Bind-Silane (refer to page 52, “Preparing
Ettan DALT preparative gels”).
For detailed information consult the Ettan DIGE System User Manual
(code 18-1173-17), the Ettan DALTtwelve System User Manual (code
80-6476-53) or the Ettan DALTsix System User Manual (code
80-6492-49)
Casting isocratic 2–D gels
1. For a full set of 14 gels, make up 900 ml acrylamide gel stock
solution without adding the 10% (w/v) ammonium persulfate
(APS) or 10% (v/v) TEMED, refer to page 8, “Other materials
required”. For best results filter the solution through a 0.2 µm
filter to remove dust and insoluble matter.
2. Assemble the caster on a level surface, as described in the Ettan
DIGE System User Manual (code 18-1173-17).
3. Connect one end of the feed tube to either a funnel or a
peristaltic pump. Insert the opposite end into the grommet in the
bottom of the balance chamber.
47
4. Pour 100 ml of displacing solution into the balance chamber.
5. When ready to pour the gels, add the appropriate volume of
freshly prepared APS and TEMED to the acrylamide gel stock
solution and mix.
6. Introduce the gel solution into the funnel or peristaltic pump
taking care not to admit any air bubbles into the feed tube.
7. Allow the solution to enter the caster until it is 1–2 cm below the
final desired height. Stop the flow of acrylamide and remove the
feed tube from the balance chamber grommet. Once the feed
tube is removed, the dense displacing solution will enter the
caster and force the remaining acrylamide solution into the gel
cassettes to the desired height.
8. Immediately pipette 1–2 ml of water-saturated butanol onto
each gel to create a level interface.
9. Allow the gels to polymerize for at least 3 hours at room
temperature before disassembling the caster. The gels can be
stored in an airtight container at 2­–8°C covered with 1 × SDS
electrophoresis running buffer for up to 2 weeks.
Equilibration of focused Immobiline DryStrips
Note: Prior to the second dimension separation, strips loaded
with saturation labeled samples should be equilibrated using DTT
only. The iodoacetamide equilibration step used with CyDye DIGE
Fluor minimal dyes must not be performed with CyDye DIGE Fluor
saturation dyes.
1. Remove focused Immobiline DryStrips from the first dimension
apparatus or if the strips have been frozen, allow them to warm
to room temperature.
2. Incubate each strip in 10 ml equilibration solution containing
DTT for 10 minutes with gentle agitation.
48
3. Meanwhile, prepare fresh agarose sealing solution and allow
to cool slightly. Immediately before applying the Immobiline
DryStrip to the second dimension gel, slowly pipette the molten
agarose sealing solution between the glass plates at the top of
the second dimension gel, taking care not to introduce bubbles.
Do not allow the agarose to cool or solidify.
4. Briefly rinse the Immobiline DryStrip by submerging in a
measuring cylinder of 1 × SDS electrophoresis running buffer.
5. With forceps, carefully place the Immobiline DryStrip between
the two glass plates of the gel. By convention, the acidic end of
the Immobiline DryStrip is on the left.
6. Gently position the strip so that it rests on the surface of the
polyacrylamide gel. Avoid trapping air bubbles between the strip
and gel. Handle the strips carefully to avoid damage to the first
and second dimension gels. Allow the agarose sealing solution
to solidify.
7 Load the gel plates into the Ettan DALT electrophoresis tank filled
with 1 × SDS electrophoresis running buffer (note: this running
buffer contains 0.2% SDS) refer to page 8, “Other materials
required”.
8. Program the desired run parameters into the control unit.
Running condition guidelines for 12 gels are given in Table 7.
Electrophoresis run time
Wattage per gel
16 hours
8 hours
4 hours
2W
4W
8W
Table 7. Recommended wattage settings for different gel running
times for 12 gels.
Always set the gels to run at 15°C and run until the bromophenol
blue dye front reaches the bottom of the gel.
49
9. Once the gels have run, they can be scanned immediately.
Ideally scan as soon as possible, e.g. on the same day. If the gels
are to be scanned later the same day they can be stored in 1 ×
SDS electrophoresis running buffer at ambient temperature in
the dark. Overnight storage should be at 2–8°C in the dark. Gels
scanned more than a day after running will show spot diffusion.
If 2–8°C storage is employed, gel plates must be allowed to
warm to ambient temperature prior to scanning
6.8. Preparing Ettan DALT preparative gels
Protein spots can be picked directly from separated proteins labeled
with CyDye DIGE Fluor saturation dyes. Reference markers
(code 18-1143-34) are used by the picking software to determine
the spot co-ordinates. Gels for spot picking must therefore be cast
with two reference markers under the gel and the gel has to be
bound to the glass plate to ensure that it does not deform during
the picking process. If using DeCyder Differential Analysis Software
to autodetect the white reference markers, please check to confirm
correct detection of each marker.
1. Treat the larger Ettan DALT low fluorescence glass plate with
Bind-Silane solution (refer to page 8, “Other materials required”
for recipe). Pipette 4 ml of Bind-Silane solution over the surface
of the plate and wipe with a lint free tissue until dry. Cover the
plate with a lint-free tissue and leave on the bench for 1.5 hours
for the excess Bind-Silane to evaporate.
2. Once dry, place a reference marker halfway down the left side
of the Bind-Silane treated plate, close to the spacer (without
touching the spacer) where it will not interfere with the protein
spot pattern. With a second marker, repeat this on the right side
of the plate.
50
3. When ready to pour the gel, sandwich the Bind-Silane treated
plate against an untreated glass plate. Place the glass plate
cassette in the gel caster. Silanized plates should only be
assembled immediately prior to gel pouring to avoid transfer of
Bind-Silane between glass surfaces within the cassette.
6.9. Scanning CyDye DIGE Fluor saturation
dye gels using Typhoon Variable Mode
Imager
Typhoon 9000 series Variable Mode Imager will optimally detect
signal from the CyDye DIGE Fluor saturation dyes. It provides the
sensitivity required for accurate quantitation of low-level signals.
Gels can be scanned between glass plates, preventing drying and
shrinkage, and allowing further rescanning if required.
If spots on the 2–D gel image show saturated signals (i.e. pixel value
exceeds 100 000) then quantitation may not be accurate. When
optimizing scan conditions, the maximum pixel value detected inside
the region of interest on the gel should be in the range 50 000–80 000.
To achieve this, it is recommended that a low resolution pre-scan is
run and the PMT adjusted accordingly until the maximum pixel value
falls within this range.
A few proteins may have high cysteine content and therefore
be labeled to a much greater extent than the general protein
population. These proteins will give much more intense spots than
the other proteins on the gel. If this is the case, it is possible to
scan at a higher PMT to saturate these few spots. This will enhance
the detection of the low abundance protein spots but it must be
recognized that the data from the saturated protein spots will not be
quantitative and should therefore be disregarded.
1. After switching on Typhoon Variable Mode Imager, leave to
warm up for 30 minutes before scanning.
51
2. Place the gel on the platen. Use the Gel Alignment Guides if
scanning assembled gels.
3. Select Fluorescence Acquisition Mode and select the appropriate
Setup scan parameters.
4. Select Tray setting.
5. Select scan Orientation using the Gel Orientation Guide to
ensure the correct option is chosen.
6. Select Press sample if scanning assembled gels.
7. Choose pixel size. Use 500 or 1000 µm for pre-scans and 100
µm for quantitative analytical scans.
8. Select Focal Plane, use +3 mm if imaging assembled gels.
9. Select DIGE File Naming Format to ensure that unique filenames
can be generated for each scan channel.
10. Press SCAN to start.
For further details please refer to Ettan DIGE System User Manual
(code 18-1173-17) or Typhoon User Guide (code 63-0028-31)
6.10. Image analysis using DeCyder 2-D
Differential Analysis Software
DeCyder 2-D Differential Analysis Software is a fully automated
software suite developed for detection, quantitation, positional
matching and differential protein expression analysis on images
generated using Ettan DIGE system.
For a detailed guide to using DeCyder 2-D Differential Analysis
Software, refer to DeCyder 2-D Differential Analysis Software User
Manual (code 28-9435-85). A rapid understanding of the software
and its capabilities can be obtained by working through the tutorials
provided with software.
The software comprises six modules shown in Table 8.
52
DeCyder module
Function
Image Loader:
For loading images into an Oracle database
DIA (Differential
In-gel Analysis)
Protein spot detection
Background subtraction
In-gel normalization
Gel artefact removal
Quantitation
All performed on a pair of images, from the
same gel.
BVA (Biological
Variation Analysis)
Matching of multiple images from different gels
to provide statistical data on differential protein
abundance levels between multiple groups.
Fold-change, Student’s T-test and ANOVA
values can all be obtained.
Batch Processor
Fully automated image detection and matching
of multiple gels without user interaction.
EDA (Extended Data Identify outliers or find groupings of the data
with Principal Component Analysis. Find
Analysis):
proteins with similar expression profiles, new
biological classes or regulatory pathways with
Pattern Analysis. Identify diagnostic markers
or classify unknown samples to known classes
with Discriminant Analysis
XML Toolbox
Extracts user specific data facilitating
automatic report generation.
Table 8. The six modules of DeCyder 2-D Differential Analysis
Software
Image analysis is performed using a number of complex algorithms,
which have been designed specifically for use with multiplexed 2–D
53
gel images. These algorithms form part of the built-in functionality of
the software, and are performed automatically with minimum user
intervention.
DeCyder Differential Analysis Software is compatible with Microsoft™
Windows™ XP Professional , Microsoft Windows Vista Business or
Windows Server 2008 Enterprise operating systems. It incorporates
operator-friendly graphical user interface (GUI), with pull-down
menus and toolbars. The combination of text and XML output files
means that all the data generated within DeCyder 2-D Differential
Analysis Software can be easily stored and accessed for further
investigation.
DeCyder 2-D Differential Analysis Software was developed in parallel
with the 2–D DIGE methodology and therefore all the advantages of
the technique are utilized in the software.
• The novel co-detection algorithm in the DeCyder 2-D Differential
Analysis Software takes advantage of the identical spot patterns
generated when multiple samples are resolved on the same gel.
The algorithm generates identical spot boundaries for spots on
images derived from the same gel.
• Conventional 2–D image analysis packages allow extensive user
intervention during spot detection and editing. This can lead to
subjective data analysis and may result in biased conclusions.
DeCyder 2-D Differential Analysis Software is designed to provide
automated spot detection, normalization, background subtraction,
matching and spot statistics. The spot detection algorithms have
been highly optimized to work with multiplexed fluorescently
labeled proteins and this allows a high degree of automation. This
minimizes user intervention, providing a more objective analysis of
the data.
• DeCyder 2-D Differential Analysis Software utilizes the pooled
internal standard experimental design. This allows unparalleled
54
accuracy for relative protein abundance quantitation and high
confidence in experimental conclusions. 2–D DIGE is the only 2–D
technique capable of multiplexing and therefore the only 2–D
approach which enables the use of an internal standard.
• Use of the internal standard experimental design also allows
the software to carry out gel-to-gel matching on the pooled
internal standard samples only. Thus, very similar images are
matched, increasing the user’s confidence in inter-gel matching.
As matching across internal standards is completed, the individual
sample images co-detected with each internal standard are
simultaneously matched into the dataset.
• DeCyder 2-D Differential Analysis Software in conjunction
with CyDye DIGE Fluor dyes allows the analysis of results from
experimental designs with various degrees of complexity. Studies
ranging from a simple control/treated experiment through to a
multi-condition experiment addressing multiple factors (e.g. dose
and time) can be performed in a single analysis.
55
7. Additional information
7.1. Requirements for cell lysis buffer
It is essential that the pH of the protein solution used with CyDye
DIGE Fluor saturation dyes is as close to pH 8.0 as possible. Below
pH 8.0, the labeling efficiency is reduced. Above pH 8.2, non-specific
labeling of lysine residues can occur, which can result in pI shifts on
the gel. To ensure that the pH is maintained at pH 8.0, a buffer such
as Tris should be included in the protein solution at 30 mM (HEPES,
MOPS, CHES, MES and tricine are also suitable). Failure to include
a suitable buffer will mean that the pH of the solution may deviate
significantly from pH 8.0 leading to the labeling problems described
above.
BEFORE sample labeling it is preferable to avoid the addition of
compounds with primary amine or thiol groups. Reagents such
as DTT and IPG buffer (thiols) or ampholytes (primary amines)
may compete with the proteins for the maleimide dye. These are
normally added in the sample buffer after the labeling step. If thiol
or primary amine containing compounds are essential, then their
effect on sample labeling should be investigated during the labeling
optimization experiments.
For samples with high levels of DNA and/or RNA we recommend
including 5 mM magnesium acetate in the cell lysis buffer to aid
solubilization of nucleic acids.
7.2. Protein lysate sonication
Sonication with a small (micro) probe sonicator provides the best and
most consistent method for disrupting cells for use in analyses using
Ettan DIGE system. Sonication will completely disrupt the cells and
will also shear the DNA and RNA in the cell, resulting in higher quality
2–D gels. The presence of large amounts of unsheared nucleic acids
56
can cause vertical streaking in a 2–D gel. DNase and RNase can be
added but these may appear as protein spots on the 2–D gel. This
protocol has been used to disrupt a range of cell and tissue types.
1. Clean the probe of the sonicator with 70% (v/v) ethanol and dry
thoroughly with a clean tissue.
2. Place the sample tube in a beaker of ice water to keep it cold
during sonication.
Note: If the sample is allowed to heat up in the presence of urea,
some proteins may be carbamylated which will alter the charge (pI)
of the protein, producing charge trains of protein across the gel.
3. Ensure that the sonicator microtip is suspended with its tip
well below the surface of the liquid in the sample tube, but not
touching the sides.
4. Start with the sonicator set initially at a low setting, such as 25%
power or 5 µm amplitude. Increase the sonication gradually so
that small white bubbles appear around the tip of the probe. This
is the ideal sonication level. When the bubbles appear, do not
increase power further as this will cause the protein sample to
froth. If the samples do froth, briefly microfuge them and then
continue sonicating.
5. When the sonication level has been optimized, sonicate for 20
second bursts followed by a 1 minute cooling period. Repeat this
process five times. Alternatively some sonicators have a pulse
facility which can be used to achieve the equivalent sonication
time. This process is completed when the sonicated solution is less
cloudy than the original solution.
6. After sonication, centrifuge the samples at 12 000 × g for 5 minutes
at 4°C. Transfer the supernatant to a new tube and discard the
pellet. The samples are now ready for protein quantitation or
storage at -70°C.
57
7.3. Adjustment of protein sample pH
If the pH of the protein sample is below pH 7.8 or above pH 8.2,
do not proceed with the labeling. First adjust the pH of the sample
before labeling.
In the following example the lysate pH is too low at pH 7.5 in a solution
containing 7 M Urea, 2 M Thiourea, 4% CHAPS and 30 mM Tris.
1. Make an identical lysis solution at pH 9.5, without the protein (7 M
Urea, 2 M Thiourea, 4% CHAPS, 30 mM Tris).
2. Mix increasing volumes of the new lysis solution to the protein
sample. This will increase the pH of the protein sample as more
cell lysis buffer is added. Stop when the pH of the protein sample
is at pH 8.0. Alternatively, the pH of the lysate can be increased to
pH 8.0 by the careful addition of 50 mM NaOH.
7.4. Cell and tissue types tested with CyDye
DIGE Fluor saturation labeling
The following set of tables show protocols that have been used for
a range of sample types, alongside examples of the 2–D images
obtained. Standard recommended protocols and reagents were used
unless otherwise stated. The protocols used here are not necessarily
optimal methods for these sample types but do present a useful
methodology along with an illustration of the image quality that can
be obtained in each case.
All IEF programs used finished with a low voltage (500 V) step for 48 hours.
This step was intended to maintain the focusing of the proteins after the
IEF program had completed. The number of hours spent at this voltage
varied for each sample type but was generally significantly lower than
the full 48 hours programmed into the IEF unit. Strips were removed
immediately upon completion of the IEF program. Where this was not
possible and samples were left at 500 V for more than 2 hours they were
then refocused by ramping to 8000 V over a period of 30 minutes.
58
Extraction and Labeling Protocols
First and Second Dimension Conditions
Extraction
Tissue washed with saline (0.9%),
mechanically homogenized in cell lysis
buffer (7 M urea, 2 M thiourea, 4%
CHAPS, 40 mM Tris,
pH 8.0, 1 ml per 0.1 g tissue) and
centrifuged (13 000 rpm, 10 minutes,
4°C). Pellet discarded and supernatant
used for labeling.
First dimension
pH 3-10 NL, 24 cm Immobiline DryStrips.
Ettan IPGphor IEF unit, anodic cup
loading.
50 µA per strip
1. 300 V, 3 hours, step
2. 600 V, 3 hours, gradient
3. 1000 V, 3 hours, gradient
4. 8000 V, 3 hours, gradient
5. 8000 V, 4 hours, step
Labeling
5 µg protein labeled with
4 nmol TCEP and 8 nmol dye.
Second dimension
12.5% Ettan DALT gel,
2 W per gel overnight, 15°C.
Gel Image
Cy3/Cy5 overlay for 5 µg protein labeled with CyDye DIGE Fluor Cy3
saturation dye (red) and 5 µg protein labeled with CyDye DIGE Fluor Cy5
saturation dye (blue).
Table 9. Mouse Brain
59
Extraction and Labeling Protocols
First and Second Dimension Conditions
Extraction
Serum-free medium was poured off
and cells washed twice with PBS in the
flask. Without trypsinization, cell lysis
buffer (2 M thiourea, 7 M urea, 4%
CHAPS, 40 mM Tris, pH 8.0) was added
to the flask. Cell lysate was pipetted
out and sonicated on wet ice, with
low-intensity 30 seconds pulses until
the lysate turned clear. The sample was
centrifuged (13 000 rpm, 10 minutes,
4°C), the pellet discarded and the
supernatant used for labeling.
First dimension
pH 3-10 NL, 24 cm Immobiline DryStrips.
Ettan IPGphor IEF unit, anodic cup
loading.
50 µA per strip
1. 300 V, 3 hours, step
2. 600 V, 3 hours, gradient
3. 1000 V, 3 hours, gradient
4. 8000 V, 3 hours, gradient
5. 8000 V, 4 hours, step
Second dimension
12.5% Ettan DALT gel,
2 W per gel overnight, 15°C.
Labeling
5 µg protein labeled with
2 nmol TCEP and 4 nmol dye.
Gel Image
Cy3/Cy5 overlay for 5 µg protein labeled with CyDye DIGE Fluor Cy3
saturation dye (red) and 5 µg protein labeled with CyDye DIGE Fluor Cy5
saturation dye (blue).
Table 10. HEP G2 cultured cell line
60
Extraction and Labeling Protocols
First and Second Dimension Conditions
Extraction
Tissue washed 4 x with saline (0.9%)
and mechanically homogenized in cell
lysis buffer (8 M urea, 4% CHAPS,
30 mM Tris, pH 8.0, 1 ml per 0.1 g
tissue). The supernatant was extracted
and sonicated on wet ice, with lowintensity 30 second pulses until the
lysate turned clear. The sample was
centrifuged (13 000 rpm, 10 minutes,
4°C), the pellet discarded and the
supernatant used for labeling.
First dimension
pH 3-10 NL, 24 cm Immobiline DryStrips.
Ettan IPGphor IEF unit, anodic cup
loading.
50 µA per strip
1. 300 V, 3 hours, step
2. 600 V, 3 hours, gradient
3. 1000 V, 3 hours, gradient
4. 8000 V, 3 hours, gradient
5. 8000 V, 4 hours, step
Second dimension
12.5% Ettan DALT gel,
2 W per gel overnight, 15°C.
Labeling
5 mg protein labeled with
2 nmol TCEP and 4 nmol dye.
Gel Image
Cy3/Cy5 overlay for 5 µg protein labeled with CyDye DIGE Fluor Cy3
saturation dye (red) and 5 µg protein labeled with CyDye DIGE Fluor Cy5
saturation dye (blue).
Table 11. Rat liver
61
Extraction and Labeling Protocols
First and Second Dimension Conditions
Extraction
Tissue washed 4 x with saline (0.9%)
and mechanically homogenized in cell
lysis buffer (8 M urea, 4% CHAPS,
30 mM Tris, pH 8.0, 1 ml per 0.1 g
tissue). The supernatant was extracted
and sonicated on wet ice, with lowintensity 30 second pulses until the
lysate turned clear. The sample was
centrifuged (13 000 rpm, 10 minutes,
4°C), the pellet discarded and the
supernatant used for labeling.
First dimension
pH 3-10 NL, 24 cm Immobiline DryStrips.
Ettan IPGphor IEF unit, anodic cup
loading.
50 µA per strip
1. 300 V, 3 hours, step
2. 600 V, 3 hours, gradient
3. 1000 V, 3 hours, gradient
4. 8000 V, 3 hours, gradient
5. 8000 V, 4 hours, step
Second dimension
12.5% Ettan DALT gel,
2 W per gel overnight, 15°C.
Labeling
5 µg protein labeled with 2 nmol TCEP
and 4 nmol dye.
Gel Image
Cy3/Cy5 overlay for 5 µg protein labeled with CyDye DIGE Fluor Cy3 saturation dye (red) and 5 µg protein labeled with
CyDye DIGE Fluor Cy5 saturation dye (blue).
Table 12. Rat lung.
62
7.5. Reagents tested for compatibility with
CyDye DIGE Fluor saturation labeling
Labeling efficiency should be tested using the labeling optimization
experiment (see page 30) in all the cases listed below:
• A new sample type is being used.
• The cell lysis buffer contains a reagent which hasn’t been tested
for compatibility with CyDye DIGE Fluor saturation labeling.
• The cell lysis buffer contains a reagent which has been tested
for compatibility with CyDye DIGE Fluor saturation labeling, but
is being used in a range known to affect labeling efficiency or is
being used outside the recommended concentration range.
• The cell lysis buffer contains a combination of reagents that
may or may not have been tested for compatibility with CyDye
DIGE Fluor saturation labeling. The effect of different reagents on
labeling efficiency is additive and may lead to unexpectedly poor
labeling when one or more interfering reagents are used together.
Reagent
Reducing agents
TCEP, Tris(2-carboxyethyl)
phosphine
Effect on CyDye DIGE Fluor saturation labeling
TCEP is used to reduce proteins before
labeling with CyDye DIGE Fluor saturation
dyes. The amount of TCEP (and dye)
that are required to label a particular
sample, are determined in the labeling
optimization experiment. If TCEP is used in
the cell lysis buffer, the optimum amount
of TCEP added for the reduction step prior
to labeling may be lower.
63
Reagent
Effect on CyDye DIGE Fluor saturation labeling
DTT,
β-mercaptoethanol
Thiol-containing reagents react with
saturation dyes. Therefore, the amount of
TCEP (and dye) may need to be increased
according to the amounts of these
compounds present in the cell lysis buffer.
The amount of TCEP (and dye) that are
required to label a particular sample, are
determined in the labeling optimization
experiment.
Detergents
CHAPS
Triton™ X-100
NP40
SDS
ASB14
Buffers
Tris
HEPES
4% recommended for use in the standard
cell lysis buffer. This can be substituted
with other detergents (see below). It is
essential when using strong detergents
(SDS, ASB14) that labeling is re-optimized
as quantification of protein concentration
may be affected.
Use up to 4% .
Use up to 4%
Use up to 0.2% - No effect on labeling.
Use up to 1% - No effect on labeling.
30–40 mM, pH 8.0 recommended. The
pH during labeling is critical. pH 7.8-8.0 is
optimal. pH >8.2 can cause non-specific
labeling.
Can cause focusing problems at high
concentrations.
64
Reagent
Effect on CyDye DIGE Fluor saturation labeling
Buffers (continued)
2-D Protein Extraction
Buffers
All Buffers are compatible with CyDye DIGE
Fluors with the following exception:
Buffer-III and -IV are not suitable when
CyDye DIGE Fluor labeling kit for scarce
samples is used since the labeling
efficiency is significantly reduced.
Protease inhibitors
Protease Inhibitor
Cocktail (Complete™),
(contains AEBSF,
4-[2-aminoethyl]benzolsulphonyl
fluoride)
Other chemicals
Amines
Ampholytes
DNAse
Compatible at manufacturer’s
recommended concentrations. To prevent
charge trains forming, a protector reagent
must be used. We recommend Pefabloc®
SCPLUS, AEBSF (Roche, code 1873601).
30 mM - 10% reduction in labeling
0.5% - No effect on labeling
1%–10% reduction in labeling
2%–20% reduction in labeling
No effect on labeling but extra spots may
be visible in 2-D gel image.
65
8. Troubleshooting guide
Problem: T
he fluorescent signal is weak when scanned on a 2–D gel.
Possible causes
Remedies
1. The CyDye DIGE Fluor
saturation dyes exceeded
their expiry date prior to
reconstitution, resulting in
poor protein labeling.
1. Check the expiry date on the
dye pack label.
2. The reconstituted dye has
been stored for too long,
resulting in poor protein
labeling. After reconstitution
CyDye DIGE Fluor saturation
dyes are only stable and
useable until the expiry date
detailed on the tube, or for 8
weeks, whichever is sooner.
2. Check the expiry date on
the dye pack label and do
not use dye that has been
reconstituted for 8 weeks or
more.
3. The DMF used to reconstitute
CyDye DIGE Fluor saturation
dyes, was of poor quality or
has been opened for longer
than 3 months, resulting in
poor protein labeling.
3. Always use 99.8% anhydrous
DMF to reconstitute CyDye
DIGE Fluor saturation dyes.
Breakdown products of DMF
include amines that compete
with the protein for dye
during the labeling step or
cause dye degradation.
4. The dyes have been exposed
to light for long periods of
time, resulting in loss of
fluorescent signal.
4. Always store CyDye DIGE
Fluor saturation dyes, in the
dark.
66
Problem: T
he fluorescent signal is weak when scanned on a 2–D gel.
Possible causes
Remedies
5. T he dyes have been left out
of the -15°C to -30°C freezer
for a long period of time,
resulting in poor protein
labeling.
5. A
lways store CyDye DIGE
Fluor saturation dyes at -15°C
to -30°C and only remove
them for short periods to
remove a small aliquot.
6. T he wrong focal plane has
been set on the Typhoon
Variable Mode Imager.
6. S
et the focal plane to
“+3 mm” for gels assembled
between standard glass
plates or “platen” for gels
placed directly on the platen.
7. T he Typhoon Variable
Mode Imager settings are
inappropriate.
7. E
nsure all parameters
comply with recommended
instrument settings.
8. T he pH of the protein lysate
is less than pH 7.8, resulting
in poor protein labeling.
This may be due to cell lysis
causing a drop in pH or
incomplete removal of the cell
wash buffer prior to addition
of the cell lysis buffer.
8. E
nsure the Tris buffer is
present at 30 mM. Increase
the pH of the cell lysis buffer
by the addition of a small
volume of 50 mM NaOH or
cell lysis buffer at pH 9.5.
9. T he pH of the protein lysate
is more than pH 8.2, resulting
in poor protein labeling.
9. Decrease the pH of the cell
lysis buffer by the addition of
a small volume of 50 mM HCl.
10. Primary amines (e.g.
pharmalytes or ampholytes)
or thiols (e.g. DTT) are
present in the labeling
reaction competing with the
10. Omit all exogenous primary
amines and thiols from the
labeling reaction.
67
Problem: T
he fluorescent signal is weak when scanned on a 2–D gel.
Remedies
Possible causes
10. Continued.
protein for dye.
11. Interfering components
are present in the labeling
reaction at too high a
concentration, resulting in
poor protein labeling.
11. Remove the compounds
from the labeling reaction
if not essential. If they are
essential test if the reduction
in labeling efficiency
can be counterbalanced
by increasing TCEP/dye
concentration. Investigate
this using the method
described in “Determining
the optimum amount of
TCEP/dye required to label a
protein lysate”, page 30.
12. There is little or no protein
in the protein lysate, or less
lysate was loaded on the
gel.
12. Test this by checking the
protein lysate concentration
using Protein Determination
Assay (USB, code 30098).
13. The protein lysate
concentration is too low i.e.
less than 0.55 mg/ml.
13. Make a new batch of
protein lysate reducing the
volume of cell lysis buffer
to increase the protein
concentration. Alternatively,
precipitate the proteins and
resuspend them in a smaller
volume of cell lysis buffer.
Always check the pH and concentration of the resuspended
sample before labeling.
68
Problem: Protein spots on the 2–D gel show MW trains and/or
streaking in the vertical direction.
Possible causes
Remedies
1. The amount of TCEP/dye used
is too low.
1. Refer back to gels from
the labeling optimization
experiment to determine
the correct amount. Repeat
labeling optimization if
necessary.
2. There is insufficient SDS in the
running buffer.
2. Prepare a fresh batch of
running buffer ensuring that
it contains 0.2% SDS.
3. The concentration of
Pharmalytes is too high.
Pharmalytes complex with
proteins at their isoelectric
point. If too much Pharmalyte
is present it may be difficult
for proteins to resolubilize
for transfer into the second
dimension.
3. Ensure that the recommended
concentration of Pharmalytes
is used (no greater than 1%
during rehydration).
Problem: Cy3 and Cy5 labeled spots for the same protein show
differential migration on the 2–D gel (i.e. some Cy3 and Cy5
labeled spots do not overlay).
Possible causes
Remedies
1. The amount of TCEP/dye used
is too low.
1. Refer back to gels from
the labeling optimization
experiment to determine the
correct amount. Repeat
69
Possible causes
Remedies
2. The sample press option
has not been selected when
scanning.
1. Continued.
labeling optimisation if
necessary.
2. Ensure that the sample
press option is used with
assembled gels.
3. Mix vigorously by pipetting
following each reagent
addition during the labeling
protocol.
3. Poor mixing during labeling,
causing non-uniform labeling.
4. Protein spots on the 2–D gel show pI charge trains and/or
streaking in the horizontal direction.
Possible causes
Remedies
1. The amount of TCEP/dye used
is too high.
1. Refer back to gels from
the labeling optimization
experiment to determine
the correct amount. Repeat
labeling optimization if
necessary.
2. Poor mixing during labeling,
causing non-uniform labeling.
2. Poor mixing during labeling,
causing non-uniform labeling.
3. The pH of the protein lysate
is above pH 8.2, resulting in
non-specific labeling of lysine
residues.
3. Check the pH of the cell
lysis buffer and reduce if
necessary by adding a small
volume of 50 mM HCl.
For further details of general 2–D electrophoresis troubleshooting,
please refer to 2–D Electrophoresis, Principles and Methods
Handbook
70
9. References
1. Ünlü, M. et al., Difference gel electrophoresis: A single gel method
for detecting changes in protein extracts. Electrophoresis 18,
2071–2077 (1997).
2. Alban, A. et al., A novel experimental design for comparative
two dimensional gel analysis: two-dimensional difference gel
electrophoresis incorporating an internal standard. Proteomics
3(1), 36–44 (2003).
3. Gruber H. J. et al., Anomalous fluorescence enhancement of Cy3
and Cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7
upon covalent linking to IgG and noncovalent binding to avidin
Bioconjug. Chem. 11 (5), 696–704 (2000).
71
10. Related products
CyDye DIGE Fluor Cy2 minimal dye (5 × 5 nmol) RPK0272
CyDye DIGE Fluor Cy3 minimal dye (5 × 5 nmol) RPK0273
CyDye DIGE Fluor Cy5 minimal dye (5 × 5 nmol) RPK0275
CyDye DIGE Fluor Cy2 minimal dye (2 × 5 nmol) 25-8008-60
CyDye DIGE Fluor Cy3 minimal dye (2 × 5 nmol) 25-8008-61
CyDye DIGE Fluor Cy5 minimal dye (2 × 5 nmol) 25-8008-62
CyDye DIGE Fluor Cy2 minimal dye (5 nmol) 25-8010-82
CyDye DIGE Fluor Cy3 minimal dye (5 nmol) 25-8010-83
CyDye DIGE Fluor Cy5 minimal dye (5 nmol) 25-8010-85
CyDye DIGE Fluor minimal labeling kit
(Cy2, Cy3 and Cy5) (5 nmol each) 25-8010-65
CyDye DIGE Fluor minimal labeling kit
(Cy2, Cy3 and Cy5) (2 nmol each) 28-9345-30
IPGbox
28-9334-65
IPGbox kit
28-9334-92
2-D Protein Extraction Buffer Trial Kit 28-9434-22
2-D Protein Extraction Buffer I 28-9434-23
2-D Protein Extraction Buffer II 28-9434-24
2-D Protein Extraction Buffer III 28-9434-25
2-D Protein Extraction Buffer IV 28-9434-26
2-D Protein Extraction Buffer V 28-9434-27
2-D Protein Extraction Buffer VI 28-9434-28
Ettan IPGphor 3 IEF system
11-0033-64
Multiphor II IEF system 18-1018-06
72
DeStreak Rehydration Solution 17-6003-19
Ettan DALTtwelve Large Vertical System
230V
115V
80-6466-27
80-6466-46
Ettan DALTsix Large Vertical System
220V
115V
80-6485-27
80-6485-08
SE 600 Ruby 80-6171-58
Low fluorescence glass plates for Ettan DALT
80-6442-14
DIGE gels 28-9374-51
DIGE Buffer Kit 28-9374-52
Reference markers
18-1143-34
Typhoon 9400 Imager 63-0055-79
Typhoon 9410 Imager 63-0055-81
Typhoon Trio 63-0055-88
Typhoon Trio+ 63-0055-90
DeCyder 2-D Differential Analysis Software 28-9435-83
Ettan DIGE System User Manual 18-1173-17
Ettan Spot Picker
18-1145-28
Ettan Digester 100/120 VAC
18-1152-59
Ettan Digester 220/240 VAC
18-1142-68
For Immobiline DryStrips please refer to the catalogue.
For product information on Typhoon Variable Mode Imager, please
inquire with your local GE Healthcare sales office.
For more details see Ettan DIGE System User Manual, catalogue and
website (www.ettandige.com).
73
GE Healthcare offices:
GE Healthcare Bio-Sciences AB
Björkgatan 30 751 84
Uppsala
Sweden
GE Healthcare Europe GmbH
Munzinger Strasse 5 D-79111
Freiburg
Germany
GE Healthcare Bio-Sciences Corp
800 Centennial Avenue
P.O. Box 1327
Piscataway
NJ 08855-1327
USA
GE Healthcare Bio-Sciences KK
Sanken Bldg. 3-25-1
Hyakunincho Shinjuku-ku
Tokyo 169-0073
Japan
For your local office contact information visit,
http://www.gehealthcare.com/lifesciences
GE Healthcare UK Limited
Amersham Place,
Little Chalfont, Buckinghamshire,
HP7 9NA UK
28953107
http://www.gehealthcare.com/lifesciences
imagination at work
25-8009-83PL AD 03/2009
Dispense
5 µg protein in
9 µl cell lysis
buffer.
Add 10 µl
TCEP at the
required
concentration*
Add
required
volume* of
2 mM TCEP.
As above
Mix vigorously by
pipetting and spin
briefly. Incubate
1 hour at 37°C,
in the dark.
Add required
volume* of
20 mM dye in
DMF**
Add required
volume* of
2 mM dye in
DMF**
** 99.8% anhydrous DMF must be used, within 3 months of opening.
Add 1 x sample buffer,
pharmalytes and DTT.
Pipette to mix. Store at
-70°C in the dark.
Warning: For research use only.
Not recommended or intended for diagnosis
of disease in humans or animals. Do not use
internally or externally in humans or animals.
As above
Add an equal volume of
2 x sample buffer.
Mix vigorously by pipetting
and spin briefly. Store at
-70°C in the dark.
25-8009-83/25-8009-84
Mix vigorously by
pipetting and spin
briefly. Incubate
30 minutes at 37°C
in the dark.
*The amount of TCEP and dye used must first be determined in labeling
optimization experiments.
Preparative Dispense
500 µg
Labeling
protein in cell
lysis buffer
Analytical
Labeling
CyDye™ DIGE Fluor saturation dye labeling
Amersham
CyDye DIGE Fluor Labeling Kit for Scarce Samples
Product protocol card
imagination at work
28-9366-83: CyDye DIGE Fluor Preparative Gel Labeling containing:
• 400 nmol CyDye DIGE Fluor Cy3 saturation dye for preparative labeling.
25-8009-84: CyDye DIGE Fluor Labeling Kit for Scarce Samples plus Preparative
Gel Labeling containing;
• 100 nmol CyDye DIGE Fluor Cy3 saturation dye for analytical labeling
• 100 nmol CyDye DIGE Fluor Cy5 saturation dye for analytical labeling
• 400 nmol CyDye DIGE Fluor Cy3 saturation dye for preparative labeling.
Ordering Information
25-8009-83: CyDye DIGE Fluor Labeling Kit for Scarce Samples containing;
• 100 nmol CyDye DIGE Fluor Cy3 saturation dye for analytical labeling
• 100 nmol CyDye DIGE Fluor Cy5 saturation dye for analytical labeling.
All chemicals should be considered as potentially hazardous. We therefore
recommend that this product is handled only by those persons who have
been trained in laboratory techniques and that it is used in accordance with
the principles of good laboratory practice. Wear suitable protective clothing
such as laboratory overalls, safety glasses and gloves. Care should be taken
to avoid contact with skin or eyes. In the case of contact with skin or eyes
wash immediately with water. See material safety data sheet(s) and/or safety
statement(s) for specific advice.
Caution: These dyes are intensely coloured and very reactive. Care should be
exercised when handling the dyes to avoid staining clothing, skin, and other
items. The toxicity of CyDye DIGE Fluor Cy3 and Cy5 saturation dyes has not yet
been evaluated.
Storage and handling
Store at -15 °C to -30 °C.
Avoid light and store in the dark.
GE Healthcare UK Limited
Amersham Place Little Chalfont
Buckinghamshire HP7 9NA UK
http://www.gehealthcare.com/lifesciences
25-8009-83PC AD 03/2009
GAll goods and services are sold subject to the terms and conditions of sale
of the company within GE Healthcare which supplies them. A copy of these
terms and conditions is available on request.Contact your local GE Healthcare
representative for the most current information.
© 2003–2006 General Electric Company – All rights reserved.
Previously published 2003
2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) technology is
covered by US patent numbers 6,043,025 and 6,127,134 and equivalent US
and foreign patents and pending patent applications and exclusively licensed
from Carnegie Mellon University. CyDye: this product or portions thereof is
manufactured under licence from Carnegie Mellon University under US Patent
Number 5,268,486 and equivalent US and foreign patents and pending patent
applications. The purchase of CyDye DIGE Fluors includes a limited license
to use the CyDye Fluors for internal research and development, but not for
any commercial purposes. A license to use the CyDye Fluors for commercial
purposes is subject to a separate license agreement with GE Healthcare.
Amersham, Biotrak and Amprep are trademarks of GE Healthcare companies.
GE, imagination at work and GE monogram are trademarks of General Electric
Company.
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