Bioscience Reports, Vol. 7, No. 3, 1987
Degradation Artefacts During Sample
Preparation for Sodium Dodecyl Sulphate
Polyacrylamide Gel Electrophoresis
E. Jane Cookson and Robert J. Beynon 1
Received April 29, 1987
KEY WORDS: gel electrophoresis; glycogen phosphorylase; protein fragmentation; western blots.
Preparation of samples for sodium dodecyl sulphate polyacrylamide gel
electrophoresis routinely involves heating the protein in solution containing detergent
and reducing agent for at least two minutes. Here we show that this treatment causes
fragmentation of the protein glycogen phosphorylase, whether purified or as a
component of a skeletal muscle preparation. The fragments are detected as minor
bands on western blots and represent the products of discrete breakage point in the
peptide sequence. Protease inhibitors cannot suppress the fragmentation.
Such small amounts of immunoreactive fragments may be incorrectly identified
on western blots as contaminants that were originally present in the antigen
preparation. They may also be a source of ambiguity in studies that search for
degradation intermediates during proteolysis.
INTRODUCTION
We report that proteins may be degraded, probably by a non-enzymic process, during
sample preparation for sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE). Such artefactual intermediates are detected readily on western blots and
we caution that care is needed in the interpretation of results obtained by this method.
Previous studies have shown that fl-galactosidase is fragmented during sample
preparation, when incubated over prolonged periods at 100~ (Kowit and Maloney,
1982). We extend these studies to demonstrate that with detection methods of
appropriate sensitivity, fragmentation can be detected over the short incubation
periods that are used routinely for sample preparation:
Department of Biochemistry, University of Liverpool, PO Box 147, Liverpool L69 3BX.
1 To whom correspondence should be addressed.
2O9
0144-8463/87/0300-0209505.00/0 9 1987 PlenumPublishingCorporation
210
Cookson and Beynon
Intracellular protein degradation requires a sequential series of proteolytic events
that result in the conversion of the native protein into its constituent amino acids. It
follows that this process might be defined by the intermediate, partially-proteolysed
forms (Beynon et al., 1985a; Wilson and Smith, 1985; Reznick et al., 1985). However,
because the levels of the intermediates are so low, detection methods are particularly
vulnerable to artefacts such as described here.
MATERIALS AND METHODS
Materials
Horseradish peroxidase-conjugated anti-mouse and anti-rabbit immunoglobulins were purchased from Dako Ltd., High Wycombe, Bucks, UK.
Nitrocellulose (pore size 0.2 #m) was obtained from Schleicher and Schuell, D-3354
Dassel, West Germany. Eupergit C (oxirane acrylic beads) was a generous gift from
Rohm Pharma GmbH, D 6108 Weiterstadt, West Germany. Highly purified rabbit
phosphorylase (obtained from Professor P. Cohen, University of Dundee) was linked
to the Eupergit beads using a published method (Hannibal-Friedrich et al., 1980).
Production of Antibodies
A polyclonal antiserum to mouse phosphorylase, purified as in Butler et al. (1984)
was raised in rabbits by subcutaneous injection of the protein (200 #g) in an emulsion
with Freund's complete adjuvant. Booster injections (200 pg in Freund's incomplete
adjuvant) were given 14, 28, 43 and 77 days later. The antibodies were affinity purified
on immobilised rabbit phosphorylase before use. A monoclonal antibody to pyridoxal
phosphate, E6(4)1, was a kind gift of Dr J. Cidlowski. The preparation and specificity
of the antibody is described elsewhere (Viceps-Madore et al., 1983). No further
purification of the ascites fluid was necessary.
Samples for Electrophoresis
Purified rabbit phosphorylase was diluted to a final concentration of I mg/ml in a
buffer consisting of 0.02 M Hepes, 0.14 M NaC1, pH 7.4. Mouse muscle soluble
proteins were obtained from hind limb and back muscles of C57BL/6J mice. Muscle
was homogenised at 4~ in 4 volumes of 0.02 M Hepes, 0.14 M NaC1, pH 7.4 and the
homogenate was centrifuged at 30,000 9/h to yield a supernatant that contained
virtually all of the glycogen phosphorylase activity. In some instances, the tissues were
homogenised in the presence of one of two mixtures of protease inhibitors:
(a) 1 mM phenylmethylsulphonyl fluoride, 10/zM E-64c and 5 mM EDTA or
(b) 150#M chymostatin, 50#M leupeptin and 5 m M 1,10 phenanthroline. The
pure phosphorylase was also treated with these inhibitors prior to electrophoresis. All
preparations of purified enzyme and mouse soluble muscle proteins were prepared for
SDS-PAGE in an identical manner. To detect phosphorylase and PLP-containing
peptides with the monoclonal antibody to the cofactor it was necessary to reduce and
thus stabilise the aldimine linkage between protein and cofactor. Protein samples were
Protein Degradation in vitro
211
treated with 160 mM NaCNBH3 for 15 minutes at 4~ in the presence of 0.25 M
imidazole citrate, pH 6.0. Unreduced samples were treated identically except for the
omission of the reductant. After reduction, the samples were mixed with an equal
volume of sample buffer (0.08 M Tris/HC1, pH 6.8, containing 2% (w/v) SDS, 0.1 M
dithiothreitol, 10% (v/v) glycerol and 0.001% (w/v) bromophenol blue) and were
placed in a 100~ water bath for 0, 2 or 10 minutes before cooling and applying to the
gel. All samples were in the presence of sample buffer for identical times before
electrophoresis.
Polyacrylamide Gel Electrophoresis
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was
performed according to the method of Studier (1973). The 1.5 mm thick gels consisted
of 12.5 % running gel and 5 % stacking gel. Electrophoresis was for 3-4 h at 40 mA at
10~ After electrophoresis, parts of the gel were stained for protein or electroblotted
into nitrocellulose. The protein stain was 0.1% (w/v) Fast Green in 45% (v/v)
methanol, 7 % (v/v) acetic acid.
Western Blotting
Proteins separated by SDS-PAGE were electroblotted onto nitrocellulose using a
modification of the method of Towbin et al. (1979). The blotting buffer was 25 mM
Tris, 192 mM glycine, 20% (v/v) methanol and 0.1% (w/v) SDS, pH 8.3. The transfer
was performed at 10~ for 20 h, using a current of 70 mA; Fast Green staining of the
gel revealed comprehensive transfer of proteins. The nitrocellulose membrane was
stained for protein using Fast Green (see above) or immunostained with anti-PLP
monoclonal antibody or affinity-purified anti-mouse phosphorylase antiserum.
Protein-bound PLP was detected on nitrocellulose using ascites fluid containing antiPLP antibody. All incubations were at room temperature with continuous shaking.
The nitrocellulose was first incubated for 1.5 h in 10 mM sodium phosphate/0.15 M
NaC1, pH 7.4, containing 0.2% (v/v) Tween 20 (PBS-T) and ascites fluid diluted
1:250,000. The nitrocellulose was then washed three times in PBS-T (5 min/wash).
Second antibody; horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin, diluted 1:1000 in PBS-T, was incubated with the nitrocellulose for 1.5 h,
followed by a further 3 washes in PBS-T (5 min/wash). Second antibody was detected
by incubating the nitrocellulose for 10 min in 100 ml PBS-T, containing 0.02 % (w/v)
diaminobenzidine and 0.03% (v/v) H202. After thorough washing in water, the
stained bands were intensified by washing in 0.5 % (w/v) CuSO4 in 0.15 M NaC1 for
5 min, prior to rinsing in water and drying in air. A similar procedure was employed for
the affinity-purified polyclonal antiserum, used at a dilution of 1:500. The second
antibody (horseradish peroxidase-conjugated pig anti-rabbit immunoglobulin) was
used at a dilution of 1:1000.
212
Cookson and Beynon
RESULTS A N D D I S C U S S I O N
The work described herein was initiated as part of an investigation of the
degradation of glycogen phosphorylase, defined in terms of the low level degradation
products that might represent breakdown intermediates in vivo (Cookson and Beynon,
1985, 1987). At an early stage in these investigations, it became apparent that the levels
of such intermediates were vanishingly low and that very sensitive methods, based on
monospecific antibodies, would be required for their detection. In contrast to many
applications based on western blotting, we required that the detection methods be
enhanced such that the parent protein was overstained and the degradation products
became visible. Thus, our approach was vulnerable to artefacts such as limited
proteolysis that might generate small but significant amounts of degradation products
in vitro. Furthermore, this was more likely to occur in heterogeneous, whole tissue
preparations than with pure phosphorylase.
Pure (recrystallised) phosphorylase was used to define the limits of the detection
systems based on the monoclonal PLP-antibody and the polyclonal phosphorylase
antiserum. We were surprised to observe that sample preparation for S D S - P A G E
resulted in a low degree of fragmentation of the protein, virtually undetectable by
Fig. 1. Fragmentation of glycogenphosphorylaseduring samplepreparation for SDS-PAGE. Highly
purified rabbit muscleglycogenphosphorylasewas treated with sodium cyanoborohydrideif required
("NaCNBH3 + "), heated at 100~ for up to 10 minutes in samplebuffer(see Methods) and separated
on SDS-PAGE (7.5/~g protein/well). Some lanes were electroblotted onto nitrocellulose and
subsequently probed using a monoclonalantibody to pyridoxalphosphate ("Anti-PLP") or an affinity
purified polyclonal antibody to phosphorylase ("Anti-phosphorylase")..A separate region of the gel,
stained for protein, is included for comparison ("Protein").
Protein Degradation in vitro
213
protein staining of the gel but readily detected by the sensitive immunochemical
techniques (Fig. 1). It is very likely that these fragments are derived from
phosphorylase as they react with both antibodies and reactivity with the P L P
monoclonal requires that the proteins are first treated with sodium cyanoborohydride
to reduce and stabilise the aldimine linkage between cofactor and protein (Butler et al.,
1985). The fragments are generated during the treatment of the protein with sample
buffer at 100~ and become more intense as the incubation time is increased from 2 to
10 minutes. Several fragments react with the polyclonal antibody but not the
monoclonal antibody. Such fragments have either lost cofactor or were derived from a
region of the polypeptide chain that did not include the PLP-binding residue Lys-680
(Johnson et al., 1987). These apo-fragments are also identifiable by their failure to shift
in mobility upon reduction with sodium cyanoborohydride. The increase in apparent
molecular weight on SDS-PAGE is a feature of phosphorylase and phosphorylasederived PLP-binding peptides that we have observed repeatedly (Butler et al., 1985;
Cookson and Beynon, 1987).
Trace contamination by proteases has the potential to cause slight fragmentation,
particularly as the substrate is denatured by a combination of SDS and an increase in
temperature and subsequently exposed in this more vulnerable conformation to the
protease (Beynon, 1987). Mixtures of protease inhibitors were added to the
phosphorylase samples prior to preparation for SDS-PAGE, but no change in banding
pattern was observed (Fig. 2). The mixtures of inhibitors were chosen as those most
Fig. 2. The effectof protease inhibitors on fragmentation of glycogen phosphorylase.A
solution of pure phosphorylasewas supplementedby a cocktail of protease inhibitors (see
Methods) beforeundergoingsample preparation for SDS-PAGE (7.5 #g protein/lane).After
separation, the proteins were electroblotted onto nitrocellulose and were probed using
antibodies to pyridoxalphosphate ("Anti-PLP") or phosphorylase("Anti-phosphorylase").
A separate region of the gel, stained for protein, is included for comparison ("Protein").
214
Cookson and Beynon
effective at suppressing muscle proteases which, in combination with the extremely
high degree of purity of the phosphorylase, renders advantitious proteolysis most
unlikely as an explanation for the fragmentation.
O u r research into phosphorylase degradation focusses on the behaviour of the
enzyme in vivo or in complex and heterogeneous tissue preparations. A high speed
supernatant from mouse skeletal muscle, containing phosphorylase (approximately
5 ~o of the total soluble protein) and other soluble muscle proteins, was also prepared
for SDS-PAGE. Extended boiling (10 minutes) causes significant fragmentation of
phosphorylase and major fragments are visible after only 2 minutes treatment (Fig. 3).
The fragments are discrete, suggesting some type of site-specific attack upon
phosphorylase. However, proteinase inhibitors were without effect on the
concentration or nature of the fragments; proteolysis can therefore be dismissed as a
cause of the phenomenon, even in crude tissue preparations. Sample preparation
requires at least a short treatment at 100~ to eliminate smearing that is otherwise
detected by the monoclonal antibody. It is noteworthy that the smearing is not due to
PLP-containing proteins because it can be observed whether or not the samples are
treated with sodium cyanoborohydride. This implies that smearing is due to nonspecific absorption of the monoclonal antibody.
Protein
Stds
Anti-PLP
97~
68--
45--
31
100~
m
....
0
2
10
10
NaCNBH3 ...............
Inhibitors ...............
_
_
+
10
0
2
10
0
2
10
+
+
+
+
+
+
+
-
+
+
+
+
+
10
Fig. 3. Fragmentation of phosphorylasein crude tissue preparations derived from mouse skeletal
muscle. A high speed supernatant from mouse skeletal muscle was prepared in the presence or
absenceof a cocktailof proteaseinhibitors (seeMethods) beforeundergoing samplepreparation for
SDS-PAGE (50#g protein/lane). After separation, the proteins were electroblotted onto
nitrocellulose and were probed using antibodies to pyridoxal phosphate ("Anti-PLP"). A separate
region of the gel, stained for protein, is included for comparison ("Protein").
Protein Degra~tation in vitro
215
The amount of protein fragmented during the preparation of samples for SDSP A G E is minute, the intensity of the parent protein band is comparable in samples of
pure phosphorylase heated for 10 minutes to untreated samples (Fig. 1). Although this
phenomenon is likely to be a characteristic of all studies involving S D S - P A G E it only
creates problems when a technique is employed which has the specificity and sensitivity
to detect the fragments. Different fragments of phosphorylase, containing PLP, are
present in the same sample of soluble mouse muscle proteins when heated for different
lengths of time (Fig. 3). A band at approximately 40,000 Mr is probably due to
transaminase(s); these appear to require at least a short incubation at 100~ in order to
form a discrete band. The band at 55,000 Mr is also enhanced by a short period of
heating, but as this band is present in all lanes it is not a PLP-protein, although it too
appears to be degraded after 10 minutes incubation. Furthermore, the fragments
produced on heating are in the size range and abundance expected of degradation
intermediates formed in vivo (Beynon et al., 1985b). Thus care is needed in the
interpretation of immuno-reactive bands, of lower molecular weight than the native
protein, as fragments derived from the intracellular degradation of the protein.
S D S - P A G E is commonly used to assess the purity of proteins and, in conjunction
with western blotting, to determine the specificity of antisera. We caution that
fragments produced during sample preparation might be misinterpreted as
contaminants.
ACKNOWLEDGEMENTS
This work was supported by grants from the Medical Research Council
(G840/7575SB) and the Muscular Dystrophy G r o u p of Great Britain (RA3/162). The
monoclonal antibody to P L P was a generous gift from Dr J. Cidlowski.
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