Enabling manual spot picking through colorimetric post

Enabling manual spot picking through colorimetric post
application note
Fluorescent protein staining
Enabling manual spot picking through
colorimetric post-staining of Deep Purple Total
Protein Stain treated 2-D gels
key words: Deep Purple Total Protein Stain • Coomassie Blue •
silver • 2-D gels • manual spot picking • MALDI ToF-Pro mass
spectrometer • dual stain
Introduction
This application note demonstrates the compatibility of
combining Deep Purple™ Total Protein Stain with
Coomassie™ Brilliant Blue or silver stains to enable manual
protein spot picking from 2-D electrophoresis gels.
Proteins in gels stained with colorimetric Coomassie Blue or
silver stain can be picked manually. Yet both stains have
drawbacks. Coomassie Blue is relatively insensitive (1), and
silver stain, while providing adequate sensitivity, offers poor
dynamic range and can be time consuming to use (2).
Although fluorescent stains are sensitive with a wide
dynamic range, they are unsuitable for manual spot picking
as they are invisible to the naked eye and require the use of
laser scanning instruments. Manual spot picking can be
performed using an ultraviolet (UV) transilluminator,
however stain signal fades with time and UV is hazardous.
Deep Purple Total Protein Stain is a highly sensitive
fluorescent stain, linear over four orders of magnitude (3, 4),
and enables good quantitation of both abundant and scarce
proteins.
When manual spot picking is required, it is therefore
attractive to consider combining the methods: to treat 2-D
gels initially with Deep Purple Total Protein Stain and then
post-stain the gels with either Coomasssie Blue or silver
stain.
Our results confirm that this combination approach
provides good, reliable protein quantitation without
sacrificing quality—no interference in mass spectrometry
analysis was observed with the dual-stained gels.
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11-0008-18 AA, 2004-06 • p1
Fig 1. A preparative gel image containing 500 µg total protein
from E. coli stained with Deep Purple Total Protein Stain.
Proteins were focused on Immobiline DryStrips and were
separated in the second dimension on Ettan DALT gels.
Products used
Amersham Biosciences products used:
Electrophoresis systems and consumables
Immobline™ DryStrip Reswelling Tray
Ettan™ IPGphor™ II IEF System
Ettan IPGphor Manifold
DALTtwelve Gel Caster
Ettan DALTtwelve Large Vertical System
Immobline DryStrip pH 3–10 NL, 24 cm
PlusOne™ Bind-Silane
PlusOne ReadySol™ IEF 40% T, 3% C
PlusOne TEMED
PlusOne Ammonium Persulfate
PlusOne Sodium Dodecylsulfate (SDS)
PlusOne Tris
PlusOne Glycine
DeStreak™ Rehydration Solution
Pharmalyte™, broad range pH 3–10
Immobiline DryStrip Cover Fluid
Trifluoroacetic Acid
80-6465-32
80-6505-03
80-6498-38
80-6467-22
80-6466-27
17-6002-45
17-1330-01
17-1310-01
17-1312-01
17-1311-01
17-1313-01
17-1321-01
17-1323-01
17-6003-19
17-0456-01
17-1335-01
17-6002-76
Fluorescent protein staining
Imaging
Typhoon™ 9410 Variable Mode Imager &
ImageQuant™ Image Analysis Software
ImageScanner™ II
63-0038-55
18-1170-84
Staining
Deep Purple™ Total Protein Stain
RPN6305
PlusOne Coomassie Tablets, PhastGel™ Blue R350 17-0518-01
PlusOne Silver Staining Kit, Protein
17-1150-01
Digestion and mass spectrometry
Ettan Trypsin, sequencing grade
Ettan MALDI-Tof Pro
17-6002-75
18-1156-53
Other materials required
Decon™ 90 (Decon Laboratories Ltd)
Hydrochloric acid
Methanol
Glacial acetic acid
Ammonia solution, puris, 25–30%
Acetonitrile
Ammonium bicarbonate
Potassium ferricyanide
Sodium thiosulphate
Recrystallized αCHCA (alpha-cyano-4-hydroxy-cinnamic acid)
(LaserBioLabs)
Crew™ wipers (Aldrich)
Methods
1-D and 2-D electrophoresis
Using the Immobiline DryStrip Reswelling Tray, 24 cm
Immobiline DryStrips pH 3–10 NL were rehydrated
overnight in DeStreak Rehydration Solution including
500 µg E. coli protein. Isoelectric focusing (IEF) was carried
out the next day using an Ettan IPGphor II IEF System with
an Ettan IPGphor Manifold. The run included three phases;
500 V for 8 h; a gradient from 500 to 4000 V for 2 h; and
8000 V for 7 h. After equilibration (5) the strips were
loaded on four large format 12.5% SDS-PAGE (Laemmli)
gels. Gel solutions were prepared using standard procedures
with PlusOne electrophoresis reagents and cast on
DALTtwelve Gel Caster with PlusOne Bind-Silane-coated
low-fluorescence glass plates (26 x 20 cm) and 1 mm
spacers. An overlay of 0.5% (w/v) agarose in running buffer
containing 0.001% bromophenol blue was applied. Seconddimension electrophoresis was performed using the Ettan
DALTtwelve Large Vertical System. Initially, gels were run at
2.5 W/gel for 30 min and then at 15 W/gel until the
bromophenol dye front reached the bottom of the gel.
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11-0008-18 AA, 2004-06 • p2
Deep Purple total protein staining and detection
After electrophoresis, the glass plates were disassembled and
the gels placed overnight in fixation solution (7.5%
[v/v] acetic acid, 10% [v/v] methanol). Staining was
performed the next day following the standard protocol (6).
After staining, the glass-backed gels were imaged using a
Typhoon 9410 Variable Mode Imager. Imaging was
performed using a 532 nm laser for excitation with a 560LP
emission filter and a photo multiplier tube set to 460 V.
Post-staining 2-D gels with Coomassie Blue and silver
After imaging, two of the gels stained with Deep Purple Total
Protein Stain were prepared for Coomassie Blue staining by
fixing them overnight in 7.5% [v/v] acetic acid and 10%
[v/v] methanol. The other two gels stained with Deep Purple
Total Protein Stain were also prepared for silver staining
through overnight fixation in 40% [v/v] ethanol and 10%
[v/v] acetic acid. Coomassie Blue staining was performed the
next day for 1.5 h according to standard protocol; gels were
then placed in a destaining solution (20% [v/v] methanol,
5% [v/v] acetic acid), with one or two solution changes until
the background was clear. Scanning was performed using the
ImageScanner flatbed scanner at a setting of 300 dpi. Silver
staining was also performed following the overnight fixation
using the PlusOne Silver Staining Kit for proteins. The silver
staining protocol was modified to make the staining
compatible with mass spectrometry (7), which requires the
omission of both glutaraldehyde in the sensitizer solution
and formaldehyde from the silver solution, while using
double the amount of formaldehyde in the developer. The
silver stain was developed for 10 min for all gels and
scanning was performed as with the Coomassie stained gels.
Mass spectrometry and protein identification
Before manual spot picking was performed, the gels were
placed in highly pure water for at least 2 h. Eight spots from
all four gels were chosen randomly and were manually
picked into microplates, and then digested with Ettan
Trypsin enzyme according to protocol (8) using the same
volumes but with modifications for the manual method.
Following digestion and extraction, the peptides were
spotted onto MALDI targets. Peptide-mass fingerprinting
was performed using Ettan MALDI-ToF Pro following the
manufacturer’s recommended protocol (9). MALDIgenerated mass spectra were internally calibrated using
trypsin peaks. The ProFound search engine (10) was used to
search peptide masses against the National Center for
Biotechnology Information non-redundant protein sequence
database (11).
Fluorescent protein staining
A
A
B
B
C
C
Fig 2. Gel dual stained with Deep Purple Total Protein Stain
and Coomassie Blue. A. Image of a gel treated first with Deep
Purple Total Protein Stain and then with Coomassie Blue.
B. Image of the same gel as in Figure 2A, but with manually
picked spots indicated. C. Mass spectrum of the spot
highlighted in Figure 2B.
Fig 3. Gel dual stained with Deep Purple Total Protein Stain
and silver. A. Image of a gel treated first with Deep Purple
Total Protein Stain and then with silver stain. B. Image of the
same gel as in Figure 3A, but with manually picked spots
indicated. C. Mass spectrum of the spot highlighted in
Figure 3B.
Results and discussion
Coomassie Blue or silver makes the gel spots visible to the
naked eye. Figure 1 shows a 2-D gel run with E. coli total
protein with Deep Purple Total Protein Stain. Figure 2 shows
gels successfully stained first with Deep Purple Total Protein
Stain and subsequently with Coomassie Blue. Eight spots per
gel were selected for manual picking (Fig 2B). Similarly, gels
initially treated with Deep Purple Total Protein Stain were
post-stained with silver, as shown in Figure 3. Eight spots
per gel were also selected for manual picking from this
collection of gels.
Colorimetric post-staining of 2-D gels
Deep Purple Total Protein Stain provides a clear, even
background that produces a high signal-to-noise ratio and
good proportionality between the fluorescent signal and
protein level. As a result, the stain is highly sensitive with
reliable quantitation. To be able to further investigate any
spots of interest using mass spectrometry when spot picking
needs to be performed manually, poststaining with
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11-0008-18 AA, 2004-06 • p3
Fluorescent protein staining
Digestion and protein identification
To verify that protein identification is not affected when gels
stained with Deep Purple Total Protein Stain are post-stained
with Coomassie Blue or silver, mass spectrometry was
performed. Figures 2C and 3C show peptide-mass
fingerprinting results for one Coomassie Blue-stained spot
and one silver-stained spot. Ninety three percent of the
proteins in the Coomassie Blue-stained plugs and 88% in
the silver-treated plugs were positively identified. In addition,
our results show that dual staining does not interfere with
mass spectrometry determination. The spectra in Figures 2C
and 3C have a low background signal, making the spectra
easy to interpret and resulting in good scores for expectation
and sequence coverage.
Conclusion
Dual staining, first with Deep Purple Total Protein Stain and
then with Coomassie Blue or silver, takes advantages of the
strengths of each stain. Deep Purple Total Protein Stain
provides high sensitivity and enables quantitation of both
abundant and scarce proteins. Post-staining gels with
Coomassie Blue or silver allows the protein spots to be
clearly seen, enabling manual spot picking. Dual staining
does not interfere with analysis by mass spectrometry and
the resulting spectra exhibit low background, facilitating easy
interpretation.
References
1.
Patton, W.F. Detection technologies in proteome analysis. J Chromatography B, 771,
3–31 (2002).
2.
Patton, W.F. A thousand points of light: the application of fluorescence detection
technologies to two-dimensional gel electrophoresis and proteomics. Electrophoresis,
21, 1123–1144 (2000).
3.
Bell, P. J. L. and Karuso P. Epicocconone. A novel fluorescent compound from the fungus
Epicoccum nigrum. J. Am. Chem. Soc., 125, 9304–9305 (2003).
4.
Mackintosh, J. A. et al. A fluorescent natural product for ultra sensitive detection of
proteins in 1-D and 2-D gel electrophoresis. Proteomics, 3, 2273–2288 (2003).
5.
Ettan DIGE User Manual, Amersham Biosciences, 18-1173-17 Edition AA (2002).
6.
Deep Purple Total Protein Stain Instructions, Amersham Biosciences, RPN6305PL
Rev-D (2004).
7.
Ettan Digester User Manual, Amersham Biosciences, 18-1167-31 Edition AB (2002).
8.
Ettan Spot Handling Workstation User Manual, Amersham Biosciences, 18-1153-55
Edition AC (2003).
9.
Ettan MALDI-ToF User Manual, Amersham Biosciences, 18-1144-01 Edition AA
(2001).
10. Genomic Solutions. ProFound-Peptide Mapping [internet]. Version 4.10.5 The
Rockefeller University Edition. New York: The Rockefeller University. [cited 2004 Feb 06]
Available from: http://129.85.19.192/profound_bin/WebProFound.exe
11. Protein [Internet]. Bethesda (MD): National Library of Medicine (US), National Center
for Biotechnology Information. [cited 2004 Feb 06]. Available from: http://
www.ncbi.nih.gov/entrez/query.fcgi?CMD=search&DB=protein
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General Electric Company reserves the right, subject to any regulatory approval if required, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice
or obligation. Contact your GE Representative for the most current information.
© 2004 General Electric Company – All rights reserved. GE and GE Monogram are trademarks of General Electric Company.
Deep Purple, DeStreak, Ettan, ImageQuant, ImageScanner, Immobiline, IPGphor, Pharmalyte, PhastGel, PlusOne, ReadySol and Typhoon are trademarks of Amersham Biosciences Limited. Deep Purple Total Protein
Stain is exclusively licensed to Amersham Biosciences from FluoroTechnics Pty Ltd. Deep Purple Total Protein Stain may only be used for applications in life science research. Amersham and Amersham Biosciences
are trademarks of Amersham plc. Coomassie is a trademark of ICI plc. Crew is a trademark of Sigma-Aldrich Inc. Decon is a trademark of Decon Laboratories Ltd.
Amersham Biosciences UK Ltd., a General Electric company, going to market as GE Healthcare.
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11-0008-18 AA, 2004-06 • p4
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