2-D Protein Extraction Buffer

2-D Protein Extraction Buffer
GE Healthcare
Data file 28-9488-39 AA
Protein sample preparation
2-D Protein Extraction Buffer
Efficient cell lysis and protein extraction are key steps for
achieving high quality results in downstream applications
such as 2-D gel analysis. 2-D Protein Extraction Buffer offers
a convenient way to prepare high quality protein lysates. The
buffers are modifications of well-studied protein solubilization
buffers, designed to produce high spot resolution for 2-D gel
analysis. Six buffers are available, supplied as a dry powder
which is rehydrated with the provided DILUENT. The optimal
buffer will depend on the nature of your sample, and
2-D Protein Extraction Buffer Trial Kit allows you to evaluate
each extraction buffer to find the most suitable buffer for
your sample (Fig 1). 2-D Protein Extraction Buffer can also be
used prior to 1-D electrophoresis, or for rehydrating IPG strips
prior to 2-D electrophoresis.
Key benefits include:
• Ready to mix buffers simplify protein extraction
preparation of cells and tissues
• 2-D Protein Extraction Buffer Trial Kit facilitates easy
optimization of total protein extraction
• Efficient and reproducible protein extraction with high yield
• Comprehensive selection of six different buffers covering
the wide range of protein diversity
• Complete declaration of buffer components, providing
transparency about what you are putting into your sample
Fig 1. 2-D Protein Extraction Buffer offers convenient and efficient protein
extraction. 2-D Protein Extraction Buffer Trial Kit includes all six extraction
buffers, allowing you to determine the most suitable extraction buffer
for your sample. 2-D Protein Extraction Buffer packs include dry buffer
component and DILUENT.
Description
Six protein extraction buffers are available, 2-D Protein
Extraction Buffer-I to -VI, which are based on urea, thiourea,
CHAPS and additional solubilizing agents as shown in Table 1.
Because the buffers are provided in a dry powder formulation,
problems associated with carbamylation are avoided.
Carbamylation, which can occur in solutions containing
urea, may alter protein charge and produce spot artifacts
on 2-D spot maps. With 2-D Extraction Buffers, buffer can
be freshly made by weighing out the necessary amount
and resuspending it in the included DILUENT. Each package
contains enough reagents necessary to prepare 50 ml
buffer, and the 2-D Protein Extraction Buffer Trial Kit includes
reagents for preparing 10 ml of each of the six buffers.
Table 1. Composition of 2-D Protein Extraction Buffer and 2-D Protein Extraction Buffer Trial Kit
2-D Protein Extraction Buffer
Composition
2-D Protein Extraction Buffer-I with DILUENT-I, 50ml
2-D Protein Extraction Buffer-II with DILUENT-II, 50ml
2-D Protein Extraction Buffer-III with DILUENT-III, 50ml
2-D Protein Extraction Buffer-IV with DILUENT-III, 50ml
2-D Protein Extraction Buffer-V with DILUENT-II, 50ml
2-D Protein Extraction Buffer-VI with DILUENT-III, 50ml
2-D Protein Extraction Buffer Trial Kit, 6 x 10 ml
Urea (< 10 M) and NP-40* (< 10%)
Urea (< 10 M) and CHAPS† (< 10%)
Urea (< 8 M), Thiourea (< 5 M), CHAPS (< 5%), and ASB-16‡ (< 5%)
Urea (< 8 M), Thiourea (< 5 M), CHAPS (< 5%), and SB 3-10§ (< 5%)
Urea (< 8 M), Thiourea (< 5 M), and CHAPS (< 10%)
Urea (< 8 M), Thiourea (< 5 M), CHAPS (< 5%), and NDSB-201¶ (< 4%)
2-D Protein Extraction Buffer-I, -II, -III, -IV, -V and –VI, including DILUENT
*
Nonylphenyl polyethylene glycol
¶
3-(1-Pyridino)-1-propane sulfonate
†
3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate ‡ Amidosulfobetaine-16 § n-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate
Applications
Compatibility with CyDye
Differential extraction of rat liver proteins
Before performing 2-D DIGE, a 1-D study was carried out
to test the compatibility of 2-D Protein Extraction Buffer
with CyDye™ DIGE Fluors. In this study, the protein lysates,
extracted using the reference buffer or the different
extraction buffers, were labeled with Cy™5 minimal dye or
Cy5 saturation dyes for scarce samples according to the
instruction manual. In these labeling reactions, 50 µg of
protein was labeled with the minimal dye and 5 µg protein
was labeled using the CyDye DIGE Fluor for scarce samples.
After labeling, the samples were analyzed by SDS-PAGE,
scanned using the Ettan™ DIGE Imager, and fluorescence
intensity was assessed. Figure 3 shows the protein samples
labeled with the minimal dye, and this figure demonstrates
that CyDye DIGE Fluor minimal dyes are compatible with
the majority of the extraction buffers, although the labeling
intensity was slightly reduced in samples extracted by
2-D Protein Extraction Buffer–I. For the saturation dyes,
a reduced labeling efficiency was observed for samples
extracted using 2-D Protein Extraction Buffer-III and –IV,
and these buffers are therefore not recommended when
saturation dyes will be used for labeling.
This study presents a comparison between different protein
extraction buffers to investigate the extraction capabilities
and differences between the buffers. Rat liver proteins
were extracted using 2-D Protein Extraction Buffer-I to -VI,
as well as a standard 2-D lysis reference buffer (8 M urea,
2 M thiourea, and 4% CHAPS). After protein extraction, the
samples were analyzed using 2-D Fluorescence Difference Gel
Electrophoresis (DIGE).
Before use, the different extraction buffers were resuspended
in the provided DILUENT according to the instructions. In
addition, the reference buffer and extraction buffers were
supplemented with 30 mM Tris-HCl (pH 8) and Protease
Inhibitor Mix (1X). Figure 2 shows the workflow, including some of
the key products that were utilized at each step. For
homogenization, the Sample Grinding Kit was used. In this
step, 0.1 ml reference buffer or 2-D Protein Extraction Buffer
was mixed with tissue samples (35 to 50 mg), in triplicate,
in tubes containing grinding resin. After grinding for 1 min
at room temperature, 0.3 ml of the same extraction buffer
was added and the samples were vortexed. The lysate was
then cleared by centrifugation (15 000×g, 20ºC, 20 min), and
the protein concentration in the cleared supernatant was
quantitated using the 2-D Quant Kit. Finally, the pH of the
lysates was adjusted to pH 8 by adding 0.5 M NaOH.
The average yield for the triplicate samples was similar for the
six different extraction buffers, ranging from 0.13–0.16 mg
protein/mg tissue. These yields were also comparable to the
reference buffer which resulted in 0.16 mg protein/mg tissue.
However, in general, 2-D Protein Extraction Buffer-III, -IV, -V,
and -VI are known to give stronger solubilization effects and
may therefore be more appropriate for harder tissues such
as muscle or membrane protein-rich cells.
2-D DIGE analysis
2-D DIGE was performed on the protein lysates extracted
with reference buffer or 2-D Protein Extraction Buffer-II to
-VI so that the resulting 2-D spot maps could be compared.
2-D Protein Extraction Buffer-I was not tested, since this
extraction buffer is not recommended when using CyDye
minimal dyes for labeling. After protein extraction, the
samples were labeled with Cy3 and Cy5 minimal dyes,
and the pooled standard was labeled with Cy2 minimal
dye, according to the instructions. For first dimension
EB-I
Tissue sample ≤ 100 mg
EB-II
EB-III
EB-IV
2-D Protein Extraction Buffer
Homogenization
Sample Grinding Kit
Quantitation of protein lysate
2-D Quant Kit
EB-V
EB-VI
Reference
Sample labeling
CyDye DIGE Fluor minimal dyes
CyDye DIGE Fluor for scarce samples
2-D electrophoresis
Immobiline DryStrip Gels
SDS-PAGE gels
Image capture
Ettan DIGE Imager
Image analysis software
DeCyder 2-D
Fig 2. Workflow using 2-D DIGE to investigate differences in extraction
capabilities of 2-D Protein Extraction Buffer-I to -VI.
2
01/2009 28-9488-39 AA
Fig 3. Rat liver samples were extracted using either the reference buffer or
2-D Protein Extraction Buffer-I to -VI. Protein extracts were subsequently
labeled with a CyDye minimal dye, and run on Excel Gel 8–18. In each set
of three lanes, 20, 10 or 5 µl of sample was loaded. Most of the extraction
buffers were compatible with minimal dyes, and gave a high labeling
intensity compared to the reference, although Extraction Buffer (EB)-I gave
a reduced labeling intensity and is therefore not recommended when using
CyDye minimal dyes.
electrophoresis, samples were run on a Multiphor™
II instrument using 24 cm Immobiline™ DryStrip Gels
(pH 3–11 NL). The strips were then loaded onto a second
dimension polyacryamide gel (12.5%). Finally, the gels were
scanned using the Ettan DIGE Imager and analyzed with
DeCyder™ 2-D Differential Analysis Software.
The results of the study showed that some spots were
differentially extracted by the buffers, which may be of
importance in maximizing yield for preparative gels and
peptide mass fingerprint identification. This also highlights
that the most appropriate extraction buffer will not only
depend on the nature of your sample, but also on the number
of protein species and which protein species that you want
to detect in your 2-D analysis. Figure 4 shows the spot map
from a sample extracted using 2-D Protein Extraction
Buffer-II overlaid on the spot map using Extraction Buffer–VI
for extraction. Several protein spots were differentially extracted
by these buffers, displayed in the figure as spots shifting
towards red (extracted to a higher degree with Extraction
Buffer-II) and spots shifting towards green (higher abundance
after extraction with Extraction Buffer-VI). The most apparent
difference between the extraction buffers was between
2-D Protein Extraction Buffer-II and the rest of the buffers,
probably because thiourea is included in Extraction BufferIII to -VI. Figure 5 shows a DeCyder software analysis
comparing the abundance of a single protein spot after
extraction with the different extraction buffers. Figure 5A
shows a protein spot that was preferentially extracted by
2-D Protein Extraction Buffer-II. Note that the thiourea-based
reference buffer gave an extraction efficiency that was
similar to Extraction Buffer-III, -IV, -V, and -VI. Figure 5B shows
a different protein spot where 2-D Protein Extraction Buffer-III
Fig 4. 2-D DIGE gel image of a protein sample extracted using Extraction
Buffer-II overlaid on a sample extracted by Extraction Buffer–VI. Protein
spots shifting towards red were extracted to a higher degree with
Extraction Buffer-II, while spots shifting towards green had a higher
abundance after extraction with Extraction Buffer-VI. The most distinct
difference between the six extraction buffers was found between
Extraction Buffer-II and the rest of the buffers.
to -VI showed slightly different extraction capabilities, with
Extraction Buffer-VI giving the highest abundance. However,
in general, the thiourea-based extraction buffers produced
only subtle differences in the resulting 2-D spot maps.
2-D Protein Extraction Buffers may also be used sequentially
in order to utilize the different extraction powers of the
buffers and maximize the amount of protein extracted from
a sample. For example, protein lysate is first made using a
urea-based buffer such as Extraction Buffer-I or -II. Sequential
A
B
Fig 5. DeCyder software analysis
showing protein abundance of
the same protein spot (shown
to the left on two different gels)
extracted using different extraction
buffers. A) The protein spot was
extracted to a higher degree with
Extraction Buffer-II. B) Another
example showing a protein spot
that gave the highest abundance
with Extraction Buffer-VI. Extraction
capability depended on the
protein spot, but in general, the
thiourea-based extraction buffers—
Extraction Buffer-III to -VI and the
reference buffer—resulted in similar
2-D spot maps.
01/2009 28-9488-39 AA
3
extractions are then carried out on the lysate, for example,
by performing a second extraction with Extraction Buffer-V
and a third extraction with either Extraction Buffer –III,
-IV, or -VI. After each extraction step, new protein spots can be
extracted and identified.
For more information regarding sample preparation solutions,
refer to Appendix I in the handbook “2-D Electrophoresis:
Principles and Methods.” In Appendix I, Section A, the
chemical components are the same components found in
2-D Protein Extraction Buffer-II (except for IPG Buffer and DTT).
In Section B, the chemical components are the same as the
components found in 2-D Protein Extraction Buffer-V (except
for IPG Buffer and DTT).
Other applications
Related products
Product
Quantity
Code no
Sample preparation
Yeast Protein Extraction Buffer Kit
28-9440-45
Mammalian Protein Extraction Buffer
28-9412-79
Sample Grinding Kit
50 samples
80-6483-37
Albumin and IgG Removal Kit
10 samples
RPN6300
2-D Quant Kit
500 assays
80-6483-56
2-D Fractionation Kit
10 samples
80-6501-04
Nuclease Mix
0.5 ml
80-6501-42
Protease Inhibitor Mix
1 ml
80-6501-23
2-D Clean-Up Kit
50 samples
80-6484-51
Related Literature
2-D Protein Extraction Buffer can also be used prior to 2-D
electrophoresis for the reswelling of Immobiline DryStrip Gels
(IPG strips) in acidic to neutral pH intervals as an alternative to
DeStreak Rehydration Solution. To simplify reswelling, IPGbox
can be used to rehydrate up to twelve IPG strips in individual
slots in a minimum volume of solution.
2-D Electrophoresis: Principles and Methods
80-6429-60
Ettan DIGE System User Manual
18-1173-17
Ordering information
Product
Quantity
Code no
2-D Protein Extraction Buffer-I
For 50 ml
28-9435-23
2-D Protein Extraction Buffer-II
For 50 ml
28-9435-24
2-D Protein Extraction Buffer-III
For 50 ml
28-9435-25
2-D Protein Extraction Buffer-IV
For 50 ml
28-9435-26
2-D Protein Extraction Buffer-V
For 50 ml
28-9435-27
2-D Protein Extraction Buffer-VI
For 50 ml
28-9435-28
2-D Protein Extraction Buffer Trial Kit
For 6 x 10 ml
28-9435-22
For local office contact information, visit
www.gelifesciences.com/contact
GE Healthcare Bio-Sciences AB
Björkgatan 30
751 84 Uppsala
Sweden
www.gelifesciences.com/sampleprep
GE, imagination at work, and GE monogram are trademarks of General
Electric Company
Cy, CyDye, DeCyder, Ettan, Immobiline, and Multiphor are trademarks of
GE Healthcare companies.
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 2 (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.
CyDye: This product or portions thereof is manufactured under an exclusive license
from Carnegie Mellon University under US patent number 5,268,486 and equivalent
patents in the US and other countries.
The purchase of CyDye products includes a limited license to use the CyDye
products for internal research and development but not for any commercial
purposes. A license to use the CyDye products for commercial purposes is subject
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If you require a commercial license to use this material and do not have one, return
this material unopened to GE Healthcare Bio-Sciences AB, Bjorkgatan 30, SE-751 84
Uppsala, Sweden and any money paid for the material will be refunded.
2-D Protein Extraction Buffer and 2-D Protein Extraction Buffer Trial Kit are
manufactured by G-Biosciences, USA.
All third party trademarks are the property of their respective owners.
©2009 General Electric Company—All rights reserved.
First published Jan. 2009
All goods and services are sold subject to the terms and conditions of sale of the
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for the most current information.
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28-9488-39 AA 01/2009
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