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(Click links below to see some alternate protocols from other facilities, the ABRF discussion group, etc.):
1. ABRF In Gel Digestion Protocol
2. UCSF In Gel Digestion Protocol
3. ABRF Discussion Thread about In-gel digestion for Mass Spec
4. Mass Spec directly from electroblot membranes
5. Passive elution of whole proteins from SDS-PAGE gels for subsequent Mass Spec analysis
a. Staining and excising spots
b. Extraction Method A - extraction into solvent for ~25 pmol or more protein per spot
c. Extraction Method B - extraction into MALDI matrix for <<25 pmol protein per spot
d. Alternate Extraction Method
In-gel Digestion protocols
Representative
ABRF In Gel Digest Protocol (copied
from ABRF Archives at www.abrf.org)
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Kenneth Williams (Kenneth.Williams@yale.edu)
Thu, 06 Nov 1997 13:24:05 -0500
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In reponse to a query posted by Katheryn Resing - this is the
representative in gel digest protocol that will accompany samples
that are
being distributed by the Internal Protein Sequence Committee. We
would
certianly welcome comments on the following protocol.
Ken Williams
________________________
ABRF Internal Protein Sequence Research Committee
Representative "In-Gel" Digestion Protocol for Proteins
in SDS PAGE Gel Slices
(10/97)
Samples to be digested in the gel are run in as few lanes as
possible to
maximize the concentration of the protein within the bands of
interest.
The gel is stained in 0.1% Coomassie R250/20% MeOH / 0.5% AcOH,
and then
destained in 30% MeOH until the bands are visible and the
background is
nearly clear. Volumes and reagent quantities described herein are
for one
band from a 1 mm gel slice digested with trypsin. A reduction and
alkylation step is included. Alternatively, the sample may be
reduced and
alkylated prior to electrophoresis. Note that an alternate buffer
system
is also provided for LysC digestion.
1. Wash the gel slices for at least 1 hr in 500 ml 100 mM
NH4HCO3.
Discard the wash.
2. Add 150 ml 100 mM NH4HCO3 and 10 ml 45 mM DTT. Incubate at
60oC for 30
min.
3. Cool to room temp and add 10 ml of 100 mM iodoacetamide and
incubate
for 30 min in the dark at room temperature.
4. Discard the solvent and wash the gel slice in 500 ml of 50%
acetonitrile/100 mM NH4HCO3 with shaking for 1 hr. Discard the
wash. Cut
the gel into 2-3 pieces and transfer to a 200 ml eppendorf style
PCR tube.
5. Add 50 ml of acetonitrile to shrink the gel pieces. After
10-15 min
remove the solvent and dry the gel slices in a rotatory
evaporator.
6. Re-swell the gel pieces with 10 ml of 25 mM NH4HCO3 containing
Promega
modified trypsin (sequencing grade) at a concentration such that
a
substrate to enzyme ratio of 10:1 has been achieved. (If the
amount of
protein is not known, add 0.1-0.2 mg of modified trypsin in 10 ml
of 25 mM
NH4HCO3.) After 10-15 minutes add 10-20 ml of additional buffer
to cover
the gel pieces. Gel pieces need to stay wet during the digest.
Incubate 4
hrs to overnight at 37oC.
Proceed to step 8 if further extraction of the gel is desired
(recommended)
- otherwise continue with step 7.
7. 0.5 ml of the supernatant may be removed for MALDI analysis
and/or the
supernatant acidified by adding 10% TFA to a final concentration
of 1% TFA
for injection onto a narrow- or microbore reverse phase column.
(If
necessary the sample's volume may be reduced ~1/3 on a rotatory
evaporator.)
8. Extraction (Optional)- Save supernatant from step 7 in tube X,
and
extract peptides from gel twice with 50 ml of 60% Acetonitrile/
0.1% TFA
for 20 min. Combine all extracts in tube X (using the same pipet
tip to
minimize losses), and speed vac to near dryness. Reconstitute in
20 ml of
appropriate solvent. Proceed with chromatography or MALDI
analysis.
Alternate Digestion Protocol.
If a LysC digest is desired a different buffer system is used,
using the
same volumes as before. This buffer system (below) has been used
successfully with LysC from Achromobacter lyticus. LysC from
other sources
has not been tested.
Wash the gel pieces in 0.5 M TrisHCl pH 9.2/50% acetonitrile.
Digest the protein(s) in the gel slice with LysC in 0.1M TrisHCl
pH 9.2.
UCSF In Gel
digestion Protocol (copied from
UCSF page at http://donatello.ucsf.edu/ingel.html
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This is the current procedure for In-Gel digests used at UCSF (last modified 8/31/2006). It has been refined to its current state by contributions from lab members over the last few years.
In-Gel Digest Procedure
For low-level proteins (<1 pmol), especially those separated by 1-D SDS-PAGE, reduction and alkylation is recommended. These procedures are performed after step 6.
Extraction of Peptides
ABRF In-Gel
Digests & Stains thread - an unedited
discussion of various issues of in-gel digestion
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JOHN@hoffman.mgen.pitt.edu (John Hempel) This compiles related
replies re. stains, and details on in-gel Lys C digestions. -JH :
From: SMTP%"Ken_Mitchelhill@muwayf.unimelb.edu.au"
10-NOV-1995 01:43:05.61 Ken Williams asked: > Has anyone
carried out comparative testing or know of any references >to
studies dealing with the relative yields of peptides obtained
from in >gel digests as a function of protein stain. Has
anyone >experience/references to results obtained with in gel
digests carried out >on proteins stained with Amido Black
and/or other stains besides >Coomassie Blue R-250? Ken,
although you asked for details of formal studies (of which I have
seen none) our anecdotal experience with hundreds of in-gel
digests is that the cleanest chromatograms come from proteins
stained with Coomassie Blue G-250 and NOT Coomassie Blue R-250.
That was when we digested the stained band, now that we destain
the band with acetonitrile/ammoniun bicarb, I don't know that the
same observation applies but we still insist on the G-250 stain.
Regards to all.......Ken Ken Mitchelhill John Holt Protein
Structure Laboratory St. Vincent's Institute for Medical Research
41 Victoria Parade, Fitzroy 3065 Australia Voice (03) 9288 2497
Fax (03) 9416 2676 ken_mitchelhill.medicine@muwayf.unimelb.edu.au
From: SMTP%"Struktur@licr.uu.se" 10-NOV-1995
13:30:56.17 Dear Ken, Ken and others, I've made 392 in gel
digests over the last 3 years on samples from our Institute and
many others, where I can't control which Coomassie was used.
However, we use R250 most of the time. Whenever there is a good
blue band, I get result; in other words I don't feel that the
yield is affected. Background peaks may vary; therefore a digest
of a not completely destained gel piece gives the non-peptide
peaks. R250 elutes sharply during my wash of the SMART C2/C18 2.1
mm column, via 10 minute gradient from 40 to 80 % acetonitrile.
Using multiple wavelength detection, we usually disguise
junk-peaks (they often contain a high proportion of aromatic
absorbance. Furthermore, as a measure of yield, I have now and
then re-digested (following the protocol in my AB paper) the same
gel piece and never found extra material, so it feels like dig
and extraction are complete! Ulf Hellman Ludwig Institute for
Cancer Research phone 46 18 550188 Fax 46 18 506867
From: SMTP%"Roland_S_Annan%notes@sb.com" 10-NOV-1995
19:14:02.22 Ken Williams, Jim Vath at Genetics Institute has
routinely been doing in-gel digests followed by MALDI from silver
stained gels. Mathias Mann at EMBL is also doing this routinely
now. We have tried it here and also found that it works, but we
don't use it routinely, yet. Carlos Fernandez-Patron has
published a several papers on reverse staining procedures using
imidazole-zinc and in one (Anal Biochem. 224, 203-211, 1995) has
performed in-gels digests on these bands. We have not tried this.
We will soon be trying Stains-All and copper stain (also reverse
stain) in the not to far future. I will let you know how it turns
out. Roland Annan Research Mass Spectrometry SmithKline Beecham
From: SMTP%"PROTCHEM@serverdos.cigb.edu.cu" 11-NOV-1995
19:47:49.74 To: Bryan Dunbar (P. Fowler), Ken Williams and all.
Hello! Here, the Protein Chemistry lab at the Center for Genetic
Engineering and Biotechnology in Havana. We have developed a high
sensitive procedure for the detection of proteins on gels based
on the principle of negative staining (Lee, Dzandu, zinc and
copper...). The new procedure (Imidazole-SDS-Zinc staining,
Reverse Staining) is significantly more sensitive than Coomassie
blue, and is specially conveived for further microanalysis or
activity, as it does not modify the protein at all (as evidenced
by MS and other procedures). Reverse Staining consists on the
formation of an imidazole-SDS-zinc 2 complex in the gel matrix,
deep white and insoluble, except in the areas where proteins are,
which remain transparent, unstained. We currently use it and it
works well at the low picomole range. As long as the gel is
stained, proteins do not difuse. (We sequenced a protein that was
kept in the RS gel for two years). In order to get the protein
amenable to further analysis (on gel digestion, blotting,
elution...) they should be "mobilized" by incubating
the gel in a zinc chelating solution. It can be applied to detect
Coomassie blue undetected proteins (i.e. those below the
detection limit of CB or those with low affinity for CB).
Detailed protocols are published in:
BioTechniques vol 12 no.4, 564-573 (1992) (the principles of the
method and applications)
Febs vol 296, 300-304 1992 (extension to gels without SDS,
increase in sensitivity, applications)
Anal. Biochem. 224, 203-211 (1995) (strategy for protein
purification based on single band transfer from RS gels,
applications)
Anal. Biochem. 224 263-269 (1995) (double staining of CB-stained
gels by imidazole-SDS-zinc RS: sensitive detection of CB
undetected proteins.)
Electrophoresis vol 16, 911-929 (1995) (single step
electrotransfer of reversed stained proteins on gels onto
reversed phase matrix) (a direct link between gel separation and
HPLC by blotting onto a reversed phase cartridge).
We will highly appreciate the comments of all users in order to
further develop these procedures. Lila Castellanos-Serra Head
Prot. Chem. Dept. Division of Physical Chemistry CIGB Havana Fax:
053 7 33 60 08 053 7 21 80 70 email
protchem@serverdos.cigb.edu.cu padron@serverdos.cigb.edu.cu
From: SMTP%"Struktur@licr.uu.se" 16-NOV-1995 18:40:14.
We store Coomassie stained gelpieces in Eppendorf tubes, just as
the gel pieces come from the destained gels, i. e. without any
liquid. We are even ignorant enough to store them at RT,
sometimes for months, and have had no problems so far. Should
they happen to get dry - no worries! Storing in liquid have at
least the potential risk that the protein might "swim
out". And why would you make a tryptic digest when Lys C
(WAKO stuff) works so excellent?? Fewer and longer fragments! We
have almost totally abandoned trypsin; we use it only for
comparative peptide maps. Lisa Bibbs and Gary Hathaway asked for
my protocol on using LysC for in gel digestion. Here it goes:
1. Wash the gelpiece(s) twice with 0.5 M TrisCl pH 9.2/50 %
acetonitrile at 30 centigrades for ca 45 min. Discard washings.
2. Dry completely using a gentle stream of nitrogen or SpeedVac.
3. Add to the dry gel 0.5 microgram of LysC in 10 microliter of
0.1 M TrisCl pH 9.2.
4. Continue adding 0.1 M TrisCl pH 9.2 in aliquotes until gel has
fully reswollen.
5. Incubate at 30 centigrades overnight.
6. Save liquid in fresh Eppy.
7. Add to the gel ca 150 microliter (dep on gel volume) 0.1 %
TFA/60 % acetonitrile. Incubate at least 60 min. Repeat and
combine extracts into the Eppy in #6 above.
8. Reduce volume (and acetonitrile!) by SpeedyVac. We remove ca
2/3.
9. Isolate fragments by narrow-bore RPC. We use the "=B5RPC
C2/C18 SC 2.1/10= " in Pharmacia'a "SMART",
utilizing a very shallow gradient (0.25 %/min).
LysC alone elutes somewhere after 40 % acetonitrile. We seldom
see any autodigestive products; at least we have NEVER sequenced
any! I hope you will be happy with this protocol; we have used it
for about 9 months now. Compared to trypsin, the yield is at
least as good, if not better. We have never seen any
"wrong" cuts; therefore we dare to "add" a
Lys BEFORE every peptide sequence we determine. And usually we
see the last Lys in peptides.
Ulf Hellman Group Head Assoc. Member Ludwig Institute for Cancer
Research Box 595 S-751 24 Uppsala Sweden Phone 46 18 550188 =46ax
46 18 506867 E-mail Ulf.Hellman@LICR.uu.se
------------------------------------------------------------------------
Created: 951220 Last Modified: 960218
MassSpec:MALDI from
blots
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>Does somebody know a method or have some suggestions, how to
do
>MALDI on proteins blotted to PVDF membranes and CBB-stained.
Arne,
Dr Christoph Eckerskorn is one of the best authorities on this
subject. His
email is eckerskorn@genmic.biochem.mpg.de (Christoph Eckerskorn).
>From his talks, I think you will find it is bad news to stain
the blot at
all. It is best to keep it wet and locate the protein by staining
a side
strip. A literature search for Eckerskorn and F. Lottspeich will
produce
appropriate papers, but here's a recent one
TI: ULTRAVIOLET MATRIX-ASSISTED LASER-DESORPTION IONIZATION-MASS
SPECTROMETRY OF ELECTROBLOTTED PROTEINS
AU: SCHREINER_M, STRUPAT_K, LOTTSPEICH_F, ECKERSKORN_C
JN: ELECTROPHORESIS, 1996, Vol.17, No.5, pp.954-961
AB: Direct mass spectrometric analysis of proteins electroblotted
onto
polyvinylidene fluoride membranes after sodium dodecyl sulfate-
polyacrylamide gel electrophoresis is demonstrated by
matrix-assisted
laser desorption ionization-mass spectrometry (MALDI-MS) with a
linear time-of-flight instrument, equipped with a nitrogen laser
(337
nm). The blotted proteins were desorbed directly from the
blotting
membrane after incubation with sinapinic acid as matrix.
Different
commercially available membranes resulted in high quality protein
signals for hydrophobic membranes exhibiting high specific
surface
areas (Immobilon PSQ or Trans-Blot) of for charged membranes
(Immobilon CD). Systematic investigations with standard proteins
were
performed to compare standard preparation procedures for
ultraviolet
(UV) MALDI-MS on stainless steel sample stages and preparation of
proteins immobilized onto membranes either by direct application
from
protein solutions (spotting) or by electrotransfer from gels
(electroblotting). Aspects such as mass resolution,
reproducibility
from shot to shot and spot to spot, mass accuracy, and
preservation
of protein localization are addressed in this paper
Good luck
Len
******************************************************
Dr Len C. Packman
Assistant Director of Research
Protein and Nucleic Acid Chemistry Facility
Department of Biochemistry, Tennis Court Road, Cambridge, CB2
1QW, UK
Tel: +44 (1223) 333639 (including answerphone)
FAX: +44 (1223) 333345
e-mail: lcp2@mole.bio.cam.ac.uk
Visit my WWW page at
http://www.bio.cam.ac.uk/proj/adr/PNAC/pnac.html
Elution of proteins from SDS-PAGE gels for subsequent Mass Spec
from Cohen,
S. L. and B. T. Chait (1997). “Mass spectrometry of whole
proteins eluted from sodium dodecyl sulfate- polyacrylamide gel
electrophoresis gels.” Anal Biochem 247(2): 257-67.
Gel Staining
and Excision
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Immediately following electrophoresis, the gels can be rinsed in
water, although we recommend against rinsing when handling
low-molecular-mass proteins (<20 kDa) that are at low picomole
gel-loading amounts. Protein bands were visualized by soaking the
entire gel in 45 µl copper-staining solution (supplied 10x from
Bio-Rad) for 5 min with gentle rocking (22). Alternatively,
zinc-staining (23) or imidazole-zincstaining (24) protocols can
be used. After staining, the gels were thoroughly rinsed in
deionized water. Because copper and zinc staining are negative
stains, protein bands appear translucent and are best viewed
against a black background. Bands with as little as 17 ng (~ 1
pmol) of myoglobin or recombinant leptin were visualized by
copper staining.
Gel Excising and Destaining (Fig. 1)
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Destaining was performed on
individual gel slices containing a single protein band as
depicted in Fig. 1 above.
Figure 1, step 1. Protein bands were excised from the stained gel
with a clean, sharp straight-edge razor (Fig. 1). The gel slices were
excised as close as possible to the boundaries of the protein
band. The resulting blue-tinted gel slices typically had the
dimensions 1 to 1.5 mm x 6-7 mm and were best handled with
flatend tweezers. Protein extraction should be performed
immediately after destaining. If a delay is anticipated, it is
best to leave the gel slices stained and stored moist (but not
soaking) at 4°C in a sealed microtube until needed.
Step 2. The destaining process completely removes the
visualization stain as well as the SDS detergent (22). Each gel
slice was treated in a separate 1.5-ml microcentrifuge tube using
standard copper-destaining protocols (Bio-Rad). Zinc-stained gels
can also be destained using the same protocols. Destaining was
performed by a three-step soaking process using a copper
Tris-glycine destain solution (Bio-Rad). The first soaking of the
gel is carried out in a solution consisting of 100 µl destain
buffer and 900 µl water. The tube is sealed and vortexed (Tomy
MT-360 36-sample microtube mixer; Tomy Tech, Palo Alto, CA) for 5
min at room temperature. During the first destaining period the
solution turns pale translucent blue (for copperstained gels) and
may become slightly foamy. The gel will retain a faint blue tint.
The second soaking is performed in fresh solution of 100 µl
destain and 900 µl water with 10 min of vortexing. At the end of
the second soaking, the destain solution and gel will be nearly
colorless. The final soaking is performed in a solution of 50 µl
destain and 950 µl water with 5 min of vortexing. At the end of
the third soaking, the destain solution as well as the gel are
clear. The destained gel pieces are rinsed in deionized water and
are ready for extraction.
Step 3. Prior to protein extraction, the gels are crushed. Before
crushing, the destained gel slices are gently blotted free of any
excess water clinging to the gel with a lint-free tissue, a step
that facilitates crushing of the gel. With the aid of tweezers,
the gel slices are placed into a 0.65-ml polypropylene
microcentrifuge tube (PGC Scientifics, Gaithersburg, MD). The
microtube should be of high quality, free of plasticizers and
contaminants that can compromise high sensitivity MS
measurements. From our experience, we avoid the use of colored
microtubes. The gel slice is manually crushed at the bottom of
the microtube for a few seconds with a sharp-pointed dental tool.
Mechanical homogenization of the gel can be used instead of the
manual crushing, but we recommend against it since the resulting
small gel pieces tend to clog pipettor tips.
At this point, either of two methods described below (Extraction
Methods A or B) can be used to extract the protein.
Extraction
Method A (Fig. 1)
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This method of extraction elutes protein directly into an aqueous
solution and is best suited to gel loadings exceeding 25 pmol.
Solution compositions that we have examined for protein
extraction include formic acid/ water/2-propanol (1:3:2 v/v/v)
(FWI), formic acid/water (1:5 v/v), water/2-propanol (2:1 v/v),
and pure water. Optimal extraction efficiency occurred with the
FWI combination, whereas no extraction was observed with pure
water. (The relative extraction efficiencies are based on a
qualitative assessment of the MALDI-MS signal-to-noise ratios).
Other acids and water-miscible organic solvents (e.g., 0.1% TFA
and acetonitrile) can be used for extraction.
Figure 1, step 4a. Sufficient extraction solution (~30-40 µl)
is added to the crushed gel to completely cover the gel pieces.
Step 5a. The tube is closed and vigorously shaken or vortexed at
room temperature from 4 to 8 h. Although not essential for
protein elution, shaking/vortexing facilitates protein
extraction. Generally, the greater the protein loading or the
smaller the protein, the shorter the vortexing time required for
extraction.
Step 6a. After the vortexing period, the microtube is centrifuged
and the supernatant is retrieved with a 10-µl micropipettor,
avoiding taking up any of the gel pieces. The crushed gel is
washed once with an equal volume of fresh extraction solution and
the wash is combined with the supernatant.
Steps 7a and 8a. If the protein is to be analyzed by MALDI-MS,
the extraction solution is lyophilized and a MALDI matrix
solution (2 µl for Fig. 2) is added to redissolve and retrieve
the protein for MS analysis using the dried-drop method of matrix
crystallization (see below). The matrix solution is FWI saturated
with 4-hydroxy-a-cyano-cinnamic acid (4HCCA) (25). If the protein
is to be analyzed by ESI-MS, the extraction solution is injected
into a protein-desalting cartridge (Michrom BioResources, Inc.,
Auburn, CA) that is mounted to the ESI capillary inlet. The
loaded cartridge is flushed several times with a 2% acetic acid
wash solution. Following the washing, the protein is eluted from
the cartridge into the electrospray source with a solution
consisting of 70% acetonitrile/2.5% acetic acid at 6 µl/min.
ESI-MS was performed on a TSQ-700 triple quadrupole (Finnigan
Corp., San Jose, CA) using standard ESI conditions for analyzing
proteins (26).
Extraction
Method B (Fig.
1)
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This method of extraction elutes protein from gels directly into
a MALDI-MS matrix solution and is best suited to gel loadings
below 25 pmol of protein or for proteins that fail to passively
elute using Extraction Method A (described above). In Method B
the matrix is allowed to undergo slow crystallization (18) during
the protein extraction period. The matrix used throughout was
4HCCA, although sinapinic acid can also be used. The moderately
low solubility of these cinnamic acid derivatives in the matrix
solution is an important characteristic for efficient slow
crystallization (18). The most frequently used matrix solution in
our experiments consisted of a saturated solution of 4HCCA in
FWI. Other solvent systems were also examined (e.g., 0.1%
TFA:acetonitrile, 2:1 v/v) (25). Gel extraction is as follows:
Figure 1, step 4b. After crushing the destained gel (see above),
enough matrix solution (~30-40 µl) is added to cover the gel
pieces.
Step 5b. The tube is closed and vigorously shaken or vortexed at
room temperature for 1-2 h.
Step 6b. Matrix crystallization is induced by leaving open the
top of the microtube (0.5-1 h) with vortexing. The matrix
solution will become slightly cloudy with a suspension of 4HCCA
crystals. An aliquot (1 µl) of the milky supernatant can be
immediately retrieved and deposited onto the MALDI probe for MS
analysis while the remaining sample is closed and left vortexing
for additional extraction. Avoid taking up any gel pieces while
pipetting and avoid crushing the matrix crystals once they are on
the probe.
Steps 7b and 8b. If the MALDI-MS response from the
"immediately retrieved" crystals is weak, the signal
should improve from crystals that have undergone a longer period
(~4-24 h) of vortexing in the gel/matrix suspension. During
extended vortexing periods, the matrix crystals precipitate to
the bottom and/or cling to the sides of the microtube and can be
retrieved with a micropipettor for MALDI-MS analysis. Also,
during the vortexing period volume losses of the solution due to
evaporation and aliquot sampling may occur. To offset the volume
losses, small amounts (5-10 µ) of matrix-free extraction
solution are added to keep the gel pieces completely submerged in
solution. For the results given in this paper vortexing times
ranged from 6 h (Fig. 5, see original paper) to 12-19 h (Fig. 6,
see original paper).
Alternate
Method - Elution of Whole Proteins
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L. Castellanos-Serra (protchem@cigb.edu.cu)
Mon, 18 May 1998 09:28:57 EST
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Dear Amanda
Working at the low picomol level we get high yields by this
passive elution procedure: Stain the gel by negative staining
(Cu, Zn or for higher sensitivity, ImH-Zn). Chelate the metal
(this is crucial!) by 2 x 5 min. incubation in Tris, 0.2-0.4M
Gly, pH 8.0 (about 1 ml per band). Wash a few seconds with 1 ml
water. Crush the gel band through a metal sieve placed in a 1 ml
syringe, to get about 32 micrometer particles. Incubate with
moderate vortexing, 2 x 10 min in Tris-Gly or ammonium
bicarbonate (30-50 mM, pH 8.0, do not add detergent at all) and 1
x 0.5 min in water. Collect each time by centrifugation ( a few
seconds). Each time the elution volume should be about twice the
volume of the crushed gel. Note that this procedure does not work
with CB stained bands, only with negative stain. Proteins reduced
in bME before electrophoresis tend to reaggregate on gel causing
low elution yields; thus, load in a sample buffer without bME or
fully reduce and alkylate the sample in 8M urea before
electrophoresis. (Ref. J. Prot. Chem., 16, 415-419, 1997,
Electrophoresis, 17, 1564-1572, 1996)
Lila Castellanos-Serra
<protchem@.cigb.edu.cu>
Prot. Chem. Dept.
Div. Phys. Chem. CIGB
FAX: 537-218070
Havana Cuba
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