Table Of ContentTECHNIQUES IN
PROTEIN CHEMISTRY III
Edited by
Ruth Hogue Angeletti
Albert Einstein College of Medicine
Bronx, New York
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Foreword
My first graduate student learned quickly that if you want to draw a
huge crowd around your poster at a meeting, don't show your latest
results from an investigation of a biological problem, but present a hot
new technique applicable to many problems. Advances in understand
ing biological systems draw the specialists, admittedly the most impor
tant people in your field; but making it possible for anyone to do
something more quickly, at higher sensitivity and accuracy, and with
less input of supplies and labor draws everyone.
I suppose that this is the wrong lesson for a graduate student to learn;
solving the problem is the objective after all. But we are always pressing
the limits of our assays, and each advance in sensitivity and speed
opens new fields of research which were previously inaccessible. Thus,
scientists will always be looking for better ways of doing things, will
always cite useful technique papers more than those advancing their
field of research (Oliver Lowry, where would we be without you?), and
will always be interested in books reporting the latest in methodology
This is the fourth volume resulting from the Protein Society sympo
sia. The number of copies sold and the desire of investigators to con
tribute to these volumes (our editors always comment on the
willingness of the authors to submit their manuscripts on time!) are in
deed impressive. The Protein Society has always devoted a lot of atten
tion at its meetings to new methodologies, and the Fifth Symposium in
Baltimore, June 22-26,1991, from which this book was compiled, was
no exception. There were more posters than ever at the symposium, and
their quality was outstanding. We are grateful for that continuing inter
est and will commit ourselves to fostering it in any way possible.
Special thanks are due Ruth Hogue Angeletti and her associates for
editing this volume. Their diligence and the top quality work presented
at the Fifth Symposium make this a most useful and informative book.
Mark Hermodson
President
The Protein Society
xiii
Preface
The pleasant surprise in editing this volume has been the enthusiastic
cooperation of the members of the Protein Society. Without this sup
port, it would have been impossible to produce a book whose ideas
are still fresh and current at publication. This active participation indi
cates to me that the Techniques in Protein Chemistry volumes have truly
become our forum for exchanging new methods and research ap
proaches and reflect the spirited exchanges of the poster sessions.
My special appreciation is due those who made this book possible:
the associate editors John Crabb, Steve Carr, Phil Andrews, and Ira
Ropson; the special reviewers Al Smith and Ed Nieves; the staff of Aca
demic Press; the presidents of the society Finn Wold and Mark
Hermodson; my secretary Elyse Rizzo for her organization and atten
tion to detail; Jim Sacchettini, who created the cover graphics; and the
authors, who did the real work while tolerating my gentle, but persis
tent nagging. I hope each volume published becomes used and
"dogeared."
Ruth Hogue Angeletti
XV
Acknowledgments
The Protein Society acknowledges with thanks the following organizations who through
their support of the Society's program goals contributed in a meaningful way to the fifth
annual symposium and thus to this volume.
AAA Laboratory
Amgen, Inc.
Applied Biosystems, Inc.
AVIV Associates, Inc.
Beckman Instruments, Inc.
Boehringer Mannheim Corporation
Dionex Corporation
Hewlett-Packard
Immuno-Dynamics, Inc.
Merck, Sharp & Dohme Research Laboratories
Millipore Corporation
Molecular Dynamics, Inc.
Monsanto Co.
National Biomedical Research Foundation
PE/SCIEX
PerSeptive Biosystems
Pharmacia LKB Biotechnology
Pickering Laboratories, Inc.
Polygen Corporation
Proton Instruments, Inc.
Rainin Instrument Co., Inc.
Savant Instruments, Inc.
Vestec Corporation
Vydac, The Separations Group, Inc.
xvii
Protein and Peptide Recovery
from PVDF Membranes
Susan Wong, Allan Padua and William J. Henzel
Department of Protein Chemistry, Genentech, Inc.
South San Francisco, CA 94080
I. Introduction
Electroblotting proteins from gels onto solid supports is a widely utilized
technique for the microisolation of proteins. Several electroblotting membranes
have been developed that allow direct sequence analysis (1-3). Polyvinylidene
difluoride (PVDF) is the most frequently utilized support for sequence analysis
of electroblotted proteins (4). A limitation of this technique is that N-terminal
blocked proteins must be chemically cleaved or proteolytically digested to
generate internal peptide fragments which can then be sequenced. In situ
digestions on PVDF (5-8) and nitrocellulose membranes (9) have been
described; however, these methods only result in partial digestion of the
membrane-bound proteins. Methods that directly extract protein from PVDF
have been developed (10,11), but are based on solutions containing detergents
which can limit subsequent proteolytic digestions or interfere with HPLC
separations. We have developed a simple extraction procedure using the solvent
dimethyl sulfoxide (DMSO) that allows recovery of intact proteins from PVDF
membranes. The DMSO can be easily removed, allowing the extract to be
analyzed by protein sequencing, amino acid analysis, or by other biochemical
analysis.
The extracted protein can be cleaved or digested and the resulting fragments
can either be separated by HPLC or by tricine SDS-gels (12) followed by a
second round of electroblotting. An additional advantage of this technique is
that the protein may be directly digested in the extraction solvent after addition
of an appropriate buffer.
Π. Materials and Methods
Materials. Proteins were purchased from Sigma, with the exception of human
growth hormone (hGH) which was obtained from Genentech, Inc. Lys-C was
from Wako chemicals, and cyanogen bromide was from Pierce. Dimethyl
sulfoxide (spectrometric grade) was from Aldrich. PVDF (Immobilon-P)
membranes were obtained from Millipore and ProBlott membranes were from
Applied Biosystems.
TECHNIQUES IN PROTEIN CHEMISTRY III
Copyright © 1992 by Academic Press, Inc.
All rights of reproduction in any form reserved. 3
4 Susan Wong et al.
Electroblotting and elution. SDS-PAGE was done as described by Laemmli
(13) using a BioRad minigel apparatus. Tricine minigels were obtained from
No vex. Electroblotting was done in a BioRad transfer tank using 10 mM 3-
[cyclohexylamino]-l-propanesulfonic acid, 20% methanol, pH 11.0 for 1 h at
250 mA (4). Coomassie stained bands from electroblotted proteins were
extracted with 200 μΐ DMSO in a micro Eppendorf tube on a VWR vortexer-2
using a medium setting for 2 h. The DMSO was removed and another 200 μΐ of
DMSO was added. The extraction was continued for an additional 2 h. The
extracts were then combined and used for further experiments. Extractions were
also performed using a single addition of 200 μΐ of DMSO, vortexed for 17h.
Fragmentation. Extracted proteins were dried in a Speed-Vac to remove DMSO
and were cleaved with cyanogen bromide in 0.1 N HC1 at 45eC for 3 h (14).
Blots were pre wetted with 5 μΐ of methanol prior to the addition of 0.1 N HC1
and cyanogen bromide.
Protease digestions were performed on electroblot extracts after partial
removal of DMSO by Speed-Vac evaporation. Ammonium bicarbonate (0.1 M)
was added to the residual DMSO (20-50 μΐ) resulting in a final solution of 200-
500 μΐ of 10% DMSO. Digestion with Lys-C was carried out at 37eC for 17 h.
with an enzyme to substrate ratio of 1:20 (w/w).
Electroblots were first treated with a modified methanol-chloroform
precipitation (15) prior to DMSO elution in order to remove Coomassie blue and
HPLC artifact peaks. Blots (wet) were placed in 100 μΐ of water in an
Eppendorf tube and 400 μΐ of methanol was added. The solution was vortexed
for 2 min. and 100 μΐ of chloroform was added. The solution was vortexed again
for 2 min. and the liquid was removed. The membrane was then extracted with
DMSO. This method effectively removed the Coomassie blue and minimized
artifact peaks in the HPLC chromatograms.
Peptide separation. Peptides were purified by reversed-phase HPLC using a
Synchropak 4000 Ä C4 column (2 x 100 mm) on a Hewlett-Packard 1090 M
liquid Chromatograph equipped with diode array detector. Peaks were detected
at 214 nm after elution with a linear gradient of 0-70% acetonitrile in 0.1%
trifluoroacetic acid at a flow rate of 200 μΐ per minute.
Protein sequencing. Automated Edman degradation was performed with
Applied Biosystems models 470A and 473A protein sequencers equipped with
on-line PTH analyzers. Sequence interpretation was performed on a VAX 8750
as described (16).
Amino acid analysis. Extracted proteins were dried in a Speed-Vac to
completely remove DMSO. Residual DMSO can result in the oxidation of some
amino acids including methionine and cysteine. Blots were prewetted with 5 μΐ
of methanol prior to hydrolysis. Proteins were hydrolyzed in the gas phase for
24 h with 300 μΐ 6N HC1 containing 0.1% phenol at 110°C under vacuum using
a Millipore Picotag workstation. Hydrolyzed blots were extracted twice with
100 μΐ of 6 N HC1. The extracts were dried and reconstituted with Beckman
sample buffer and hydrolysates were analyzed on a Beckman Model 6300 amino
acid analyzer.
Protein/Peptide Recovery from PVDF Membranes 5
ΠΙ. Results and Discussion
In an effort to optimize recovery, a number of proteins were transferred to
Immobilon-P or ProBlott membranes, stained with Coomassie and then
extracted for various times with DMSO. The optimal extraction time was found
to be 4 hours using 2 consecutive 2 hour extractions (of 200 μΐ DMSO each) or
a single extraction of 200 μΐ for 17 h. Both extraction methods resulted in
nearly identical protein recovery. Recovery values obtained from DMSO
extractions of proteins on ProBlott and Immobilon-P extractions are shown in
Table I. These values were obtained by using the 4 hour extraction procedure.
The extraction efficiency from Immobilon-P was better than from Problott
membranes. Other extraction solvents were examined including 50% mixtures
of DMSO and methanol, and DMSO and water (data not shown). These
solvents resulted in lower efficiency of extraction of protein from PVDF
membranes compared to using DMSO alone.
Extraction efficiencies can be determined by analyzing an aliquot of the
extract by amino acid analysis. Proteins which are found to be intractable to
DMSO extractions can be cleaved with cyanogen bromide and the resulting
peptides can then be efficiently extracted. Further purification of these peptides
can be accomplished by SDS-PAGE or reversed-phase HPLC. Extraction of
cyanogen bromide peptides from the membrane may be a preferred alternative
to directly extracting intact proteins larger than 70 kDa. No indication of
selective retention of hydrophobic peptides was seen when membranes were
sequenced after DMSO extractions. The extraction efficiencies of cyanogen
bromide cleaved proteins are shown in Table II. Peptide recovery from PVDF
and ProBlott membranes were similar.
TABLE I. Recovery data of proteins eluted from PVDF electroblots.
Proteins were separated on 12% SDS gel, electroblotted onto
Immobilon-P or ProBlott and stained with Coomassie brilliant blue.
Bands were excised and extracted with DMSO, transferred to hydrolysis
tubes, and analyzed by amino acid analysis. Extracted blots were
hydrolyzed to determine residual protein. Protein on the blot was
calculated by adding the amount remaining on the blot after extraction to
the extracted amount. All numbers represent the average of triplicate
experiments.
Amount Protein Protein Percent
on gel on blot eluted recovery
Protein Membrane (pmol) (pmol) (pmol) (±SD)
Carbonic Immobilon-P 100 45 31 69 ±1
anhydrase ProBlott 100 44 31 70±7
hGH Immobilon-P 225 80 68 85 ±10
ProBlott 225 110 84 76±6
Lysozyme Immobilon-P 210 56 42 75±7
ProBlott 210 89 39 44 ±9
BSA Immobilon-P 108 50 18 36 ±1
ProBlott 108 38 12 32 ±1
6 Susan Wong et al.
TABLE II. Recovery of peptides eluted from PVDF electroblots
following CNBr cleavage. Proteins bound to PVDF membranes were
cleaved with CNBr in 0.1 N HC1 at 45°C, dried, then extracted with
DMSO. The DMSO extracts were transferred to hydrolysis tubes and
analyzed by amino acid analysis. All numbers represent the average of
duplicate experiments.
Protein Protein Percent
on blot eluted recovery
Protein Membrane (pmol) (pmol) (±SD)
Carbonic Immobilon-P 61 55 90 ±2
anhydrase ProBlott 50 45 90 ±5
BSA Immobilon-P 40 22 55 ± 1
ProBlott 18 10 56 ±4
The application of peptide mapping on proteins eluted from electroblots is
shown in Figure 1. Human growth hormone was eluted from a ProBlott
membrane. An aliquot was removed (25%) and analyzed by amino acid
analysis, which indicated a total of 72 pmol was originally present in the extract.
The remaining 54 pmol was cleaved with cyanogen bromide, dried in a Speed-
Vac, electrophoresed on a 16% tricine SDS gel and electroblotted on a ProBlott
membrane. The excised bands were sequenced by automated Edman
degradation and revealed initial yields ranging from 2-16 pmol (Fig. 1).
A BC DE
kDa
tew —97
mm -66
•a** —45
-31
: 215
β*^''-21~ =s
*" J§g^g|||f§§^ '„,' 4HMHI —14.4 —14
— -8.2
-6.2
-2.5
Initial
yield
Band (pmoP
5 10 15 20
1 2.5 FPTIPLSRLFDNAMLRAHRL
2 2.0 LRAHRLHQLAXDTYQE
3 16.0 GRLEDGSPRTGQIFKQTYSKFDT
Figure 1. Coomassie blue stained ProBlott membrane of CNBr cleaved hGH after DMSO
elution from a ProBlott membrane. (Lane A) Recombinant hGH was electrophoresed on a 12%
SDS gel, electroblotted onto ProBlott, stained with Coomassie blue, the band excised and
extracted with DMSO. The extracted protein (54 pmol) was cleaved with cyanogen bromide,
and the peptides separated on a 16% tricine SDS gel. Bands 1-3 were sequenced (A);
(B) control, hGH (100 pmol) + CNBr; (C) control, hGH (100 pmol); (D) LMW marker proteins
(BioRad); (E) LMW marker (Diversified Biotech).
Protein/Peptide Recovery from PVDF Membranes 7
A B C D E F GH I JK
8.2-
6.2-
,..,,. ^igeiiiPÄi^ii II^^^^*#S:
2.5-
Figured SDS-PAGE tricine gel (16%) of a Lys-C digest of hGH. Lane A is
intact hGH, Lane B is a Lys-C digest of hGH, Lanes C-F are Lys-C digests of
hGH with increasing amounts of DMSO added from 10 to 40% in 10%
increments, Lane G is a Lys-C digest of hGH with 0.1% SDS. Lane H-K are
the same conditions as in C-F except 0.1% SDS was included in the digestion
buffer. All digestion were performed in 0.1M NH4HCO3 containing 0.01M
DTT.
Proteins extracted from PVDF are difficult to resolubilize in aqueous buffers
if DMSO is completely removed. To circumvent this problem proteins were
digested with Lys-C in the presence of DMSO. Figure 2 shows the results of
digesting hGH in the presence of increasing amounts of DMSO. Addition of
DMSO to the digestion had little effect on proteolysis as shown in lanes C-F.
The addition of detergents such as SDS can be used to solubilize hydrophobic
proteins and denatured proteins. The effects of increasing amounts of DMSO on
the digestion of hGH by Lys-C in 0.1% SDS, 0.1 M ammonium bicarbonate is
shown in lanes H-K in Fig. 2. DMSO significantly enhanced the activity of
Lys-C in the presence of SDS (lanes H-K) resulting in greater proteolysis than
seen with SDS alone (lane G) or in the absence of DMSO and SDS (lane B).
Higher amounts of DMSO (30-40%) (lanes J-K) result in a similar cleavage
pattern as observed when hGH is digested in the absence of SDS (lanes B-F). A
similar effect with dimethylformamide was recently reported (17).
TABLE ΙΠ. Sequence analysis obtained from an electroblot
of a Lys-C digest of hGH eluted from a ProBlott membrane.
Initial
yield
M.W. (pmol)
5 10 15 20 25
4K 49 FPTIPLSRLFDNAMLRAHRLHQLAFDTYQE
5K 5 XNLELLRISLLLIQSWLEPVQ
