Amersham™ CyDye DIGE Fluors - GE Healthcare Life Sciences

Amersham™ CyDye DIGE Fluors - GE Healthcare Life Sciences
Amersham™
CyDye DIGE Fluors
(minimal dyes) for 2D DIGE
Reagents for labeling protein with Cy2, Cy3 and Cy5 dyes,
before 2-Dimensional separation
Product Booklet
Codes:RPK0272 25-8008-60
RPK027325-8008-61
RPK027525-8008-62
25-8010-65
25-8010-82
25-8010-83
25-8010-85
28-9345-30
Page finder
1.Legal
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2. Handling 2.1. Safety warnings and precautions 2.2. Storage 2.3. Expiry
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3. Components6
4. Other materials required
7
5.Description
9
6. Protocol12
6.1. Introduction
12
6.2. Preparation of a cell lysate compatible with CyDye DIGE
12
minimal labeling
6.3. Reconstitution of CyDye in Dimethylformamide (DMF)
to give a stock solution
14
6.4. Preparation of CyDye solution used to label proteins
15
6.5. Minimal labeling a protein sample
16
6.6. Loading samples onto IPG strips
17
7.
Additional information
7.1. Requirements for 2D DIGE protein lysis buffer
7.2. Requirements for a cell wash buffer
7.3. Cell sonication
7.4. Adjustments of protein sample pH
7.5. Testing a new protein lysate for successful labeling
7.6. Post-staining with Deep Purple Total Protein Stain
7.7. Reagents tested with 2D DIGE
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8. Troubleshooting guide27
9. Related products
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2
1. Legal
GE, imagination at work and GE monogram are trademarks of
General Electric Company.
Amersham, Cy, CyDye, DeCyder, Deep Purple, Ettan, ImageQuant,
IPGphor, Immobiline, Multiphor, Pharmalyte, PlusOne and Typhoon
are trademarks of GE Healthcare companies.
2-D Fluorescence Difference Gel Electrophoresis: 2-D Fluorescence
Difference Gel Electrophoresis (2D 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.
Complete is a trademark of Roche
Sigma is a trademark of Sigma-Aldrich Co.
Prefabloc is a trademark of Pentapham Ltd.
Triton is a trademark of Union Carbide Chemicals and Plastic
Company Inc.
© 2002–2013 General Electric Company - All rights reserved.
Previously published 2002.
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.
3
http://www.gelifesciences.com
GE Healthcare UK Limited.
Amersham Place, Little Chalfont,
Buckinghamshire, HP7 9NA, UK
4
2. Handling
2.1. Safety warnings
and precautions
CAUTION: This dye is intensely
colored and very reactive. Care
should be exercised when
handling the dye to avoid
staining clothing, skin, and
other items. The toxicity of
Cy™2, Cy3 and Cy5 NHS Esters
has not yet been evaluated.
Warning: For research only.
Not recommended or intended
for diagnosis of disease in
humans or animals. Do not
use internally or externally in
humans or animals.
Warning: 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.
2.2. Storage
Store at -15°C to -30°C. Avoid
light, store in the dark.
2.3. Expiry
For expiry date see outer
packaging.
5
3. Components
RPK0272:
25 nmol (5 × 5 nmol) of CyDye™ DIGE Fluor Cy2
minimal dye
RPK0273:
25 nmol (5 × 5 nmol) of CyDye DIGE Fluor Cy3
minimal dye
RPK0275:
25 nmol (5 × 5 nmol) of CyDye DIGE Fluor Cy5
minimal dye
25-8008-60: 10 nmol (2 × 5 nmol) of CyDye DIGE Fluor Cy2
minimal dye
25-8008-61: 10 nmol (2 × 5 nmol) of CyDye DIGE Fluor Cy3
minimal dye
25-8008-62: 10 nmol (2 × 5 nmol) of CyDye DIGE Fluor Cy5
minimal dye
25-8010-65: 5 nmol of CyDye DIGE Fluor minimal labeling
kit (Cy2, Cy3 and Cy5)
25-8010-82: 5 nmol of CyDye DIGE Fluor Cy2 minimal dye
25-8010-83: 5 nmol of CyDye DIGE Fluor Cy3 minimal dye
25-8010-85: 5 nmol of CyDye DIGE Fluor Cy5 minimal dye
28-9345-30: 2 nmol of CyDye DIGE Fluor minimal labeling
kit (Cy2, Cy3 and Cy5)
6
4. Other materials required
Reconstitution of CyDye
99.8% anhydrous Dimethylformamide (DMF) less than 3 months old
from day of opening (Sigma-Aldrich 22,705-6)
Labeling
• Microfuge tubes 1.5 ml
• S
tandard Cell wash buffer, 10 mM Tris (pH 8.0), 5 mM Magnesium
Acetate. Store in aliquots at -15°C to -30°C.
• L ysis buffer 30 mM Tris, 7 M Urea, 2 M Thiourea, 4% (w/v) CHAPS.
Adjust to pH 8.5 with dilute HCl. Aliquots can be stored at -15°C
to -30°C.
• Lysine 10 mM L-Lysine (Sigma-Aldrich L-5626)
• pH indicator strips (Sigma™ pH test strips pH 4.5–10.0 P4536)
For additional protocols
• 2 × Sample buffer 8 M Urea, 130 mM DTT, 4% (w/v) CHAPS, 2% (v/v)
Pharmalyte™ 3-10 for IEF. Aliquots can be stored at -15°C to -30°C.
ehydration buffer
• R
8 M Urea, 4% (w/v) CHAPS, 1% (v/v) Pharmalyte 3-10 for IEF
13 mM DTT
odium Hydroxide
• S
50 mM NaOH
× Gel loading buffer
• 2
120 mM Tris (pH 6.8), 20% (v/v) (87% [v/v] Glycerol), 4% (w/v) SDS,
200 mM DTT and a few grains of Bromophenol Blue.
2.5% Acrylamide gel (for SE600) 32 ml Acrylamide/Bis 40% (v/v),
• 1
25 ml Tris (1.5 M pH 8.8), 1 ml SDS 10% (v/v), 1 ml Ammonium
Persulfate 10% (w/v), 40 µl TEMED. Make up to 100 ml with
distilled water.
7
• 1 × SDS electrophoresis running buffer 25 mM Tris, 192 mM
Glycine, 0.2% SDS. Store at room temperature.
8
5. Description
2D Fluorescence Difference Gel Electrophoresis (2D DIGE) is a
method to label proteins with CyDye Fluors that are subsequently
separated using 2-Dimensional gel electrophoresis. This protocol is
specifically designed to work using CyDye DIGE Fluors.
The 2D DIGE technology is designed to simplify the process of
detecting and identifying proteins using the 2-D electrophoresis
technique by allowing the separation of up to three different protein
samples in the same 2-D gel. Each of the three protein samples
are labeled with one CyDye. After labeling the three samples are
mixed together and run on the same Isoelectric focusing (IEF) and
SDS PAGE gel. The ability to multiplex different samples on the same
gel means that the different samples will be subject to exactly the
same 1st and 2nd dimension running conditions so the same protein
labeled with a CyDye will migrate to the same position on the 2-D
gel, this helps limit experimental variation.
Each of the individual samples can then be visualized independently
by selecting the individual excitation and emission wavelengths for
each CyDye when fluorescence scanning.
Experimental design
The experimental design recommended is based on evidence that
the experimental variation in a 2-D gel electrophoresis experiment
is mostly due to gel to gel variation. Running multiple samples on a
single gel reduces the number of gels required to produce the same
number of data sets. The recommended protocol suggests that an
internal standard sample be run on all gels within an experiment.
The standard sample is generated by mixing together an aliquot of
all the different samples in an experiment. This means that every
protein from every sample will be represented in the standard
that is present on all the gels. The standard sample will increase
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the confidence in matching between gels and will also allow the
generation of accurate spot statistics between gels.
When using DeCyder™ 2D software use the experimental design
outlined below. The example given is for protein samples, one
derived from a control tissue and one a diseased tissue:
Mix 1/3 of each of the control and diseased samples together to
create a standard sample. Label the standard sample with Cy2.
Label the remaining 2/3 of the control sample with Cy3.
Label the remaining 2/3 of the diseased sample with Cy5.
More complex experimental designs can be generated using the
standard sample on all gels.
Minimal labeling
CyDye DIGE Fluors have an NHS ester reactive group, and are
designed to covalently attach to the epsilon amino group of lysine
of proteins via an amide linkage. The quantity of dye added to the
sample is limiting in the reaction hence this method is referred to
as ‘minimal’ labeling. This ensures that the dyes label approximately
1–2% of the available lysine and then only on a single lysine per
protein molecule.
The three dyes are matched for size and charge such that the three
labeled protein samples are all run on the same 2-D gel the same
protein labeled with each CyDye will overlay.
The lysine amino acid in proteins carries a +1 charge at neutral or
acidic pH. CyDye DIGE Fluors also carry an intrinsic +1 charge which,
when coupled to the lysine, replaces the lysine’s +1 charge with its
own, ensuring that the pI of the protein does not significantly alter.
CyDye DIGE Fluors when coupled to the protein add approximately
500 Da to the protein’s mass in a uniform manner, giving an image
comparable to equivalent silver stained gels in existing databases.
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Spot picking
To spot pick from a gel containing CyDye labeled samples, post stain
the gel with Deep Purple to ensure that the majority of unlabeled
protein is picked to give sufficient protein for MS identification. The
migration difference between the unlabeled and labeled protein is
due to the addition of a single CyDye molecule to the protein. This is
more significant for low molecular weight proteins.
Protein identification
CyDye labeling of proteins does not affect the mass spectrometry
data used to identify proteins as only 1–2% of lysine residues are
labeled on a single protein. CyDye labeling of the lysine will only
result in the loss of a single trypsin digest site per labeled protein.
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6. Protocol
6.1. Introduction
This protocol provides all the information required to use CyDye
DIGE Fluors to label proteins for 2-D electrophoresis experiments. It
is recommended that the protocol is read thoroughly before using
the system and that it is followed precisely. Reagents tested with 2D
DIGE are listed on page 25–26.
In the standard labeling protocol, proteins are first solubilized in a
lysis buffer. The protein concentration should then be determined
using a standard protein quantitation method. CyDye DIGE Fluors
are then added to the protein lysate so that 50 µg of protein is
labeled with 400 pmol of fluor. The reaction is incubated on ice in the
dark for thirty minutes.
When handling proteins it is important to keep them on ice at all
times to reduce the effect of proteases, and use plastic tubes as
many proteins will adhere to glassware.
The fluorescent properties of Cy2, Cy3 and Cy5 can be adversely
affected by exposure to light, so it is recommended that all labeling
reactions are done in the dark, in microfuge tubes, and the exposure
of protein labeled with CyDye to all light sources is kept to a
minimum.
6.2. Preparation of a cell lysate compatible
with CyDye DIGE minimal labeling
The example given here is that used with an Escherichia coli
model system (See Ettan DIGE user manual for more details).
Recommendations for different cell types are listed on page 24.
Approximately 4 × 1010 E. coli cells will result in a 5 to 10 mg/ml of
protein in 1 ml of lysis buffer.
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1. Pellet the cells in a suitable centrifuge at 4°C.
2. Pour off all growth media, taking care not to disturb the cell pellet.
3. Resuspend cell pellet in 1 ml of standard cell wash buffer in a
microfuge tube.
4. Pellet the cells in a microcentrifuge at 12 000 × g for 4 minutes at
4°C.
5. Remove and discard the supernatant.
6. Resuspend cell pellet in 1 ml of standard cell wash buffer in a
microfuge tube.
7. Repeat steps 4 to 6 at least three times.
8. Ensure all the wash buffer has been removed with a fine pipette.
9. Resuspend the washed cell pellet in 1 ml of lysis buffer (30 mM
Tris, 7 M Urea, 2 M Thiourea, 4% (w/v) CHAPS, pH 8.5) and leave on
ice for 10 minutes.
Note: if the protein concentration is less than 5 mg/ml after protein
quantitation, resuspend cells in a correspondingly smaller volume of
lysis buffer in subsequent experiments.
10. Keep the cells on ice and sonicate intermittently until the cells
are lysed. See ‘Cell sonication’, page 19.
11. Centrifuge the cell lysate at 4°C for 10 minutes at 12 000 × g in a
microcentrifuge.
12. Transfer supernatant to a labeled tube. This is the cell lysate to
be used for CyDye labeling. Discard the pellet.
Check that the pH of the cell lysate is still at pH 8.5 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 ‘Adjustment of protein sample pH’, page 20.
Store cell lysates in aliquots at -70°C until protein concentration is
to be determined.
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6.3. Reconstitution of CyDye in
Dimethylformamide (DMF) to give a stock
solution
The dry CyDye must be reconstituted in DMF. Each vial of CyDye
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 will give a deep color; Cy2-yellow,
Cy3-red, Cy5-blue.
Displacement of CyDye during manufacture or shipment of the fluors
can be recovered to the bottom of the tube by pipetting the DMF
down the side of the tube, vortexing vigorously, and centrifuging.
The quality of the DMF used in all experiments is critical to ensure
that the protein labeling is successful. The DMF must be anhydrous
and every effort should be used 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 NHS ester CyDye reducing the concentration of fluor available
for protein labeling. If in doubt use an unopened batch of DMF for
reconstituting the fluor.
1. Take a small volume of DMF from its original container and
dispense into a fresh microfuge tube.
2. Remove the CyDye from the -15°C to -30°C freezer and leave to
warm for 5 minutes at room temperature.
3. After 5 minutes add the specified volume of DMF to each new vial
of CyDye (see specification sheet supplied with the dye). This gives
a CyDye stock solution of 1 mM. For the 2 nmol pack size, this
gives a CyDye working solution of 0.4 mM and should be used
immediately.
4. Replace the cap on the dye microfuge tube and vortex vigorously
for 30 seconds.
14
5. Centrifuge the microfuge tube for 30 seconds at 12 000 × g in a
microcentrifuge.
6. The fluor can now be used.
Unused CyDye 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 stock solution is only stable and usable
until the expiry date detailed on the tube or for 2 months, whichever
is sooner.
6.4. Preparation of CyDye working solution
used to label proteins
Note: 2 nmol pack sizes do not need to undergo this process. 1 µl of
the 2 nmol CyDye working solution contains already 400 pmol.
1. B
riefly spin down CyDye stock solution prepared in protocol 3, in
a microcentrifuge.
2. A
dd one volume of CyDye stock solution to 1.5 volumes of highgrade DMF, to make 400 µM CyDye solution. For example, take
2 µl CyDye stock solution and add 3 µl DMF to give 400 pmol
CyDye in 1 µl.
Note: Add the DMF first to the microfuge tube, followed by CyDye.
1 µl of the diluted dye now contains 400 pmol.
Quantity of CyDye to be used to label a protein lysate
It is recommended that 50 µg of protein is labeled with 400 pmol of
CyDye.
In each tube of CyDye there will be a 1 mM CyDye stock solution.
It is recommended that 400 pmol of CyDye per 50 µg of protein to be
labeled, between 100 pmol and 1000 pmol per 50 µg of protein can
be used. If labeling more than 50 µg of protein then the same fluor
to protein ratio must be used for all samples on the same gel.
15
Examples of CyDye dilutions that are used (recommended example
is highlighted) are shown in Table 1.
Table 1 - Examples of some widely used CyDye dilutions
Volume of stock Volume of added Total volume (µl) Concentration of
CyDye (µl)
DMF (µl)
CyDye (pmoles/µl)
1
4
5
200
2
3
5
400
2
2
4
500
1
-
1
1000
The CyDye working solution is only stable for 1 week at -15°C to
-30°C.
6.5. Minimal labeling a protein sample
The recommended concentration of the protein lysate is between 5
and 10 mg/ml. Samples containing from 1 mg/ml to 20 mg/ml have
been successfully labeled using the protocol below.
The amount of CyDye used in the labeling reaction will have to be
determined individually for the type of protein sample being analyzed.
1. Add a volume of protein sample equivalent to 50 µg to a
microfuge tube. Bulk labeling reactions can also be done using
more protein and dye.
2. Add 1 µl of diluted CyDye to the microfuge tube containing the
protein sample (i.e. 50 µg of protein is labeled with 400 pmol of
dye for the labeling reaction).
3. Mix and centrifuge briefly in a microcentrifuge. Leave on ice for
30 minutes in the dark.
4. Add 1 µl of 10 mM lysine to stop the reaction. Mix by pipetting and
spin briefly in a microcentrifuge.
5. Leave for 10 minutes on ice in the dark.
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6. Samples can now be stored for at least three months at -70°C in
the dark.
6.6. Loading samples onto IPG strips.
1. After the protein samples have been CyDye labeled add an equal
volume of 2 × sample buffer and leave on ice for 10 minutes.
2. Pool the protein samples that are going to be separated on the
same 1st and 2nd dimension gel.
There are now two options
Option 1 - The samples can be loaded onto a rehydrated IPG strip
via cup loading on the Multiphor™ II or IPGphor™.
Option 2 - Follow the protocol outlined below if the IPG strips are to
be rehydrated in the presence of the protein sample.
The total volume of labeled protein needs to be made up to the
volume required for each IPG strip using the rehydration buffer.
One protein sample labeled with one CyDye: 20 µl
Add an equal volume 2 × sample buffer:
20 µl + 20 µl = 40 µl
Add three samples together: 40 µl × 3
Total volume:
120 µl
A 24 cm IPG strip needs a total volume of 450 µl so add (450 µl –
120 µl) = 330 µl of rehydration buffer
Samples can now be run on an IPGphor, followed by the 2nd
dimension on an Ettan Dalt twelve or six . For instructions on
running 1st dimension IEF and 2nd dimension SDS PAGE gels,
refer to the Ettan DIGE manual or the manuals supplied with the
electrophoresis equipment.
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7. Additional information
7.1. Requirements for 2D DIGE protein lysis
buffer
It is essential that the pH of the protein solution used with a CyDye
DIGE Fluor is between pH 8.0–9.0.
To ensure that the pH remains between pH 8.0–9.0 a buffer such as
Tris, Hepes or Bicarbonate should be included in the protein solution
at a concentration of approximately 30 mM. Failure to include a
suitable buffer will mean that the pH of the solution will fall below
pH 8.0 resulting in little or no protein labeling. The Lysis buffer is
required to work at 4°C so the pH should be checked when the
solution is chilled.
The protein solution should not contain any added primary amine
compounds BEFORE labeling.
Primary amines, such as ampholytes, will compete with the proteins
for CyDye. The result will be fewer CyDye labeled proteins, which
might affect the data after scanning and spot detection.
See the section ‘Reagents tested with 2D DIGE’, page 25.
7.2. Requirements for a cell wash buffer
A cell wash buffer should not lyse the cells, but it should dilute and
remove any growth media or reagents that might affect the CyDye
labeling process.
The cell wash buffer should not contain any primary amines.
A range of cell wash solutions such as 75 mM Phosphate Buffered
Saline (PBS) can be used in conjunction with the DIGE technology
as long as their compatibility with the 2D DIGE labeling is evaluated
in controlled experiments (see ‘Testing a new protein lysate for
successful labeling’, page 20).
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7.3. Cell sonication
Sonication with a small (micro) probe sonicator provides the best
and most consistent method for disrupting cells for use in 2D DIGE.
Sonication will completely disrupt the cells and will also shear
the DNA and RNA in the cell, resulting in higher quality 2-D gels.
Presence of large amounts of unsheared nucleic acids can cause
vertical streaking in a 2-D gel. DNases and RNAses can be added
but these may appear as protein spots on the 2-D gel. Sonication
can be used on many different cell types including bacteria and
mammalian cells.
1. Clean the probe of the sonicator with 70% (v/v) Ethanol and dry
thoroughly with a clean tissue.
2. Place a beaker of ice water around the sample tube to keep it cold
during sonication.
If the sample is allowed to heat up in the presence of urea, some
proteins will 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 now at an ideal sonication level. When the bubbles appear, do
not increase the power any further as this will cause the protein
sample to froth. If the samples do froth, briefly microfuge them
and then continue sonicating with a reduced power level.
5. When the sonication is at the ideal level, sonicate for 20 second
bursts followed by a 1 minute cooling period. Repeat this process
five times. Alternatively, some sonicators have a pulse facility
19
which can be used to achieve the equivalent sonication time.
Some samples may need further sonication cycles.
6. Sonication is complete when the solution appears significantly
less cloudy than the starting solution.
7. After sonication, centrifuge the samples at 12 000 × g for 5 minutes
at 4°C. Remove the supernatant to a new tube and discard any
pellet.
8. Samples are now ready for labeling with CyDye.
7.4. Adjustment of protein sample pH
If the pH of the protein sample is below pH 8.0 do not proceed with
labeling with CyDye, first, increase the pH of the sample.
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. M
ake an identical lysis solution, (7 M Urea, 2 M Thiourea, 4%
CHAPS, 30 mM Tris [without the protein]) at pH 9.5.
ix increasing volumes of the new lysis solution to the protein
2. M
sample. This will increase the pH of the protein sample as more
lysis buffer is added. Stop when the pH of the protein sample is at
pH 8.5.
Alternatively, the pH of the lysate can be increased to pH 8.5 by
the careful addition of dilute Sodium Hydroxide (50 mM).
7.5. Testing a new protein lysate for
successful labeling
It is important to check that the labeling of the proteins has worked
before the sample is used in 2D DIGE.
The method involves running a small sample of your new-labeled
lysate on 1-D SDS-PAGE gel along with a control lysate that has
20
already been labeled successfully. This gel is then scanned for
CyDye, and the total fluorescence of each labeled sample is
compared.
1. L abel the new protein samples with Cy5 following the instructions
in ‘Protein sample labeling with CyDye’, page 16. Cy5 labeled
lysates have negligible cross talk with the Deep Purple dye that
might be used later in the experiment.
dd a volume of each CyDye labeled protein lysate equivalent to
2. A
50 µg into a microfuge tube.
dd an equal volume of 2 × gel loading buffer to the labeled
3. A
protein lysate.
4. H
eat the samples at 95°C for 5 minutes to ensure full reduction of
the proteins.
5. M
ake serial dilutions of each of the lysates in the 2 × gel loading
buffer such as 25 µg, 12.5 µg and 6.25 µg. Make a 12.5% SDSPAGE gel between low fluorescence glass plates (See Ettan DIGE
User manual for glass plate recommendations). The gel should
be made with wells at the top of the gel where the samples
will be loaded. The SE600 gel system is recommended for this
verification.
6. Load each protein serial dilution in successive lanes on the gel.
7. R
un the samples until the Bromophenol Blue dye front has nearly
reached the bottom of the gel.
can the gel at the Cy5 wavelength with Typhoon™ and carry out
8. S
the statistics in ImageQuant™ software. If the labeling efficiency
is comparable to the control, CyDye labeled samples can be
subjected to 1st and 2nd dimension electrophoresis.
9. If further labeling is required to test a chemical component then
the whole process should be repeated starting at point 1, or the
21
sample can be resuspended in the recommended lysis buffer after
using the PlusOne™ 2-D Clean Up Kit from GE Healthcare.
If labeling of the new lysate appears poor compared to the control
sample the reason for this must be determined. The pH of the
sample should be satisfactory, as this has previously been checked
with pH test strips. The simplest explanation is that less of the new
lysate was loaded on the gel than the control lysate. This can be
tested by post-staining the gel for total protein using Deep Purple, or
a general post stain such as silver staining. If an equivalent amount
of the new sample and control sample were loaded on the gel this
would suggest that there is a chemical component in the lysate that
is affecting the labeling reaction.
7.6. Post-staining with Deep Purple Total
Protein Stain
The fluorescent Deep Purple Total Protein Stain fits into the standard
2D gel electrophoresis workflow and is particularly suitable for use
with the 2D DIGE system. The recommended workflow involves
the matching of Deep Purple post-stained preparative gels with
CyDye labeled analytical gels. Deep Purple has been shown to
be compatible with the image analysis softwares and the stain
is compatible with manual or automated spot picking and mass
spectrometry for protein identification applications.
1. P
lace an appropriate volume of fixation solution into the
containers that will be used to process gels. The recommended
volume of fixation solution required is ~20 fold excess of the gel
volume (1000 ml for Ettan DALT gels).
2. Dismantle the electrophoresis apparatus. Remove one glass plate
and place the gel attached to the glass plate into fix solution.
Note: Place only one gel in each container. The Immobiline™
DryStrip can be left attached to help with gel orientation.
22
3. Incubate in the fixation solution overnight at room temperature
with gentle agitation.
4. Take the stain out of the -15°C to -30°C freezer and allow to stand
at room temperature for 5–10 minutes.
5. Pour off the fixation solution and replace with the wash solution
(1000 ml for Ettan DALT gels). Wash with gentle agitation for 30
minutes.
6. Pour off the wash solution and replace with 500 ml water. To
make up the working stain solution, briefly shake the stain
concentrate and add 2.5 ml Deep Purple to make a 1:200 dilution.
Cover the container to create a dark environment and incubate
for 1 hour at room temperature with gentle agitation.
Note: The solution is light sensitive and should be kept out of bright
light.
Note: Containers can be wrapped in foil or covered with black
plastic. It is not necessary to eliminate light completely, only to
ensure that bright light is significantly reduced. Alternatively,
containers with lids, that are a solid colored plastic, may be used.
7. Pour off the stain and replace with 7.5% (v/v) acetic acid. Cover
the container to create a dark environment and incubate with
gentle agitation for at least 15 minutes.
8. Repeat the acetic acid step once. The gel can be imaged at this
stage.
NOTE: If speed is more important than background levels, the gel
can be imaged after one acetic acid step. Further washes in acetic
acid can be performed to reduce the background still further if
necessary. After imaging, the gels can be stored in the dark in 7.5%
(v/v) acetic acid at 2–8 ºC for several weeks. This allows for further
imaging at a later date if required.
23
Table 1. Cells and tissue types tested with 2D DIGE.
Cell or tissue
type
Lysis buffer
Method of cell or tissue
disruption
Saccharomyces 2 M Thiourea
cerevisiae
7 M Urea
30 mM Tris 4%
CHAPS (pH to 8.5)
Quantity
of dye
required to
label 50 µg
protein
400 pmol
/50 µg
Pellet washed with PBS.
Cells sonicated on wet ice
and spun in a microfuge
for 5 min at 12 000 × g.
Pellet discarded and protein
concentration determined.
Escherichia coli 7 M Urea
Pellet sonicated on wet ice 400 pmol
2 M Thiourea
at amplitude 7 microns, for /50 µg
30 mM Tris 4%
30 seconds pulses. Spun
CHAPS (pH to 8.5) in a centrifuge at 4°C at
12 000 × g for 10 minutes.
Pellet discarded and protein
concentration determined.
Rat liver
7 M Urea
1 g of tissue placed in 10 ml 400 pmol
2 M Thiourea
/50 µg
of lysis buffer. Tissue then
10 mM Tris
homogenized and sonicated
5 mM Magnesium and lysate centrifuged at
Acetate
10°C, at 12 000 × g for 1
4% CHAPS (pH
hour. Pellet then discarded
to 8.0)
and protein concentration
determined.
Rat heart
7 M Urea
1 g of tissue placed in 10 ml 400 pmol
2 M Thiourea
/50 µg
of lysis buffer. Tissue then
10 mM Tris pH 8
homogenized and sonicated
5 mM Magnesium and lysate centrifuged at
Acetate
10°C, at 12 000 × g for 1
4% CHAPS (pH
hour. Pellet then discarded
to 8.5)
and protein concentration
determined.
Tomato fruit
7 M Urea
Phenol based total protein 200 pmol
wall
2 M Thiourea
/50 µg
extraction, including
4% CHAPS
homogenizing step.
10 mM Tris (pH
to 8.5)
For further examples of cell and tissue types tested with 2D DIGE please refer
to the Ettan DIGE User Manual.
24
7.7. Reagents tested with 2D DIGE
Reducing agents
DTT
2 mg/ml - slight reduction in labeling
5 mg/ml - 2 × reduction in labeling 10 mg/ml 10 × reduction in labeling CyDye will bind thiols
at increased concentrations.
TCEP tris(2-carboxyehtyl)
Phosphine
0.5 mM to 1 mM - slight reduction in labeling
2 mM - significant reduction in labeling
β-mercaptoethanol – Significantly reduces
labeling
Detergents
Triton™ X-100
use at 1%
17% reduction in labeling
NP40
up to 1%
No effect on labeling
SDS
up to 1%
Salts
Application of sample during rehydration <10 mM recommended
Application of sample via cup-loading
<50 mM recommended
Buffers
Tris
Recommend 10–40 mM pH 8.5 pH is very
important. pH 8 to 9 is optimal.
Hepes
Can cause focusing problems at high
concentrations.
Bicarbonate
5 mM pH 8.5
Acceptable
Ches
5 mM pH 9–9.5
Acceptable
PPA is a (3-piperidino
-propionamide)
5 mM pH 8
Drop in sensitivity as PPA
primary amine.
2-D Protein
Extraction Buffers.
All buffers are compatible with CyDye DIGE Fluor minimal dyes
25
Protease inhibitors
AEBSF (Pefabloc™)
(4-(2-aminoethyl)Benzolsulphonyl Fluoride)
causes charge trains unless protector
reagent is used
Protease Inhibitor Cocktail
(Complete™)
as above as contains AEBSF
Aprotinin
compatible at recommended
concentrations
APMSF (4-amidino-phenyl)
Methane-sulphonyl
Fluoride)
compatible at manufacturer’s
recommended concentrations
EDTA (Ethylene
Diaminetetra-Acetic acid)
compatible between 0.5 mM and
10 mM
PMSF
(Phenylmethylsulphonyl
compatible at manufacturer’s
recommended concentrations
Pepstatin A
compatible at manufacturer’s
recommended concentrations
26
8. Troubleshooting guide
Problem: The pH of the protein lysate is less than pH 8 prior to
labeling.
Possible causes
Remedies
1. The lysis of the cells has
caused a drop in the pH.
1. Increase the buffering
capacity of the Lysis buffer
to 40 mM Tris (50 mM is the
recommended maximum for
Tris).
2. The cell wash buffer was not
completely removed prior to
addition of the lysis buffer.
2. Increase the pH of the lysis
buffer by the addition of
a small volume of 50 mM
NaOH. Or add an equal
volume of the lysis buffer
that is at pH 9.5.
Problem: The fluorescent signal is weak when scanned on a 2-D gel.
Possible causes
Remedies
1. The fluors after reconstitution
have a fixed lifetime in
DMF that may have been
exceeded.
1. Check the expiry date on
CyDye.
2. The DMF used to reconstitute
CyDye was of poor quality or
has been opened for longer
than 3 months.
2. Always use the 99.8%
anhydrous DMF to
reconstitute CyDye DIGE
Fluors. Breakdown products
of DMF include amines which
compete with the protein for
the CyDye labeling.
3. CyDye has been exposed to
light for long periods of time.
3. Always store CyDye in the
dark.
27
Problem: The fluorescent signal is weak when scanned on a 2-D gel
continued.
Remedies
Possible causes
4. CyDye has been left out of
the -20°C freezer for a long
period of time.
4. Always store CyDye at -15°C
to -30°C and only remove
them for short periods to
remove a small aliquot.
5. The wrong focal plane has
been set on the Typhoon.
5. Set the focal plane to
“+ 3 mm” for gels assembled
between standard glass
plates or “platen” for gels
placed directly on the platen.
6. The pH of the protein lysate
is less than pH8.
6. Increase the pH of the lysis
buffer by the addition of
a small volume of 50 mM
NaOH. Or add an equal
volume of the lysis buffer
that is at pH 9.5.
7. Primary amines such as
Pharmalyte or ampholytes
are present in the labeling
reaction competing with the
protein for CyDye.
7. Omit all exogenous primary
amines from the labeling
reaction.
8. DTT or other substances
such as SDS are present in
the labeling reaction at too
high a concentration.
8. Remove the substances
from the labeling reaction
if not essential. If they are
essential test if the reduction
in labeling efficiency can
be counterbalancedby
increasing CyDye
28
Problem: The fluorescent signal is weak when scanned on a 2-D gel
continued.
Possible cause
Remedies
8. Continued.
concentration. Investigate
this using the ‘Testing a new
protein lysate for successful
labeling’, page 20.
9. There is little or no protein
in the protein lysate, or less
lysate was loaded on the
gel.
9. Test this using the ‘Testing
a new protein lysate for
successful labeling’ section
and ‘Post-staining with Deep
Purple’, page 22.
10. The protein lysate
concentration is too low i.e.
less than 1 mg/ml.
10. a. Make a new batch of
protein lysate reducing
the volume of lysis buffer
to increase the protein
concentration.
b. Increase the ratio of
CyDye to protein.
c. Precipitate the proteins
and resuspend them in
a smaller volume of lysis
buffer.
Always check the pH and
concentration of the new
sample before labeling.
29
Problem: The fluorescent signal is weak when scanned on a 2-D gel
continued.
Possible cause
Remedies
11. Incorrect fluor to protein
ratio.
11. 400 pmol of fluor per
50 µg of protein is
recommended. If there is a
large concentration of other
components which can
react with the fluor, then
more fluor (up to 2 nmol
per 50 µg of protein) can be
used.
30
9. Related products
2-D Protein Extraction Buffer Trial Kit
2-D Protein Extraction Buffer I
2-D Protein Extraction Buffer II
2-D Protein Extraction Buffer III
2-D Protein Extraction Buffer IV
2-D Protein Extraction Buffer V
2-D Protein Extraction Buffer VI
28-9435-22
28-9435-23
28-9435-24
28-9435-25
28-9435-26
28-9435-27
28-9435-28
IPGphor 3 Isoelectric Focusing Unit
11-0033-64
Multiphor II 18-1018-06
IPGbox28-9334-65
Ettan DALTsix Separation Unit 220V
Ettan DALTsix Separation Unit 115V
80-6485-27
80-6485-08
SE600 Ruby
80-6171-58
DIGE gels
DIGE Buffer Kit
28-9374-51
28-9374-52
Low Fluorescence glass plates
Ettan Dalt SE600 80-6475-58
80-6179-94
Typhoon FLA 9500 28-9969-43
DeCyder 2D 7.2 SPN license
28-9854-11
Ettan Spot Picker
Ettan Digester
18-1145-28
18-1142-68
For Immobile DryStrip gels please refer to the catalogue
For more details see Ettan DIGE user manual, catalogue and website.
31
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 Japan Corporation
Sanken Bldg. 3-25-1, Hyakunincho,
Shinjuku-ku, Tokyo 169-0073,
Japan
For your local office contact information, visit
www.gelifesciences.com/contact
GE Healthcare UK Limited
Amersham Place
Little Chalfont, Buckinghamshire,
HP7 9NA, UK
http://www.gelifesciences.com
28953163
imagination at work
RPK0272PL AG 11/2013
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