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ANALYTICAL BIOCHEMISTRY
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the addition of gelatin reduced mobility of the standards by 13 { 3.4%; in 10% T, the reduction was 16
{ 3.8%; and in 12.5% T, the reduction was 15 { 2.8%.
As this effect is consistent regardless of acrylamide
concentration, it is likely due to the added sieving
effect of gelatin polymerized into the matrix of the
gel. We have also routinely observed this same level
of retardation with nonprotease proteins from S.
commune (data not shown). In contrast, proteases migrate much more slowly in gelatin SDS PAGE than
would be predicted by the retardation of standards.
For the proteases we tested, the reduction of mobility
due to addition of gelatin in a 10% T acrylamide gel
was 48% for chymotrypsin, 44% for elastase, 43% for
trypsin, 41% for papain, and 28% for ScPrB. The degree of retardation is not constant from one protease
to another. This means that it is impossible to predict
directly what the position of a protease band will be
in a gelatin SDS gel based on its molecular weight.
In addition, it suggests that molecular weights of proteases cannot be estimated accurately in gelatin
SDS gels.
The degree of overestimation of relative molecular
weights is shown in Table 1. The magnitude of the
overestimation appears to be reduced with increasing
acrylamide concentration in the gels. This would be
expected because of the increased sieving effect of the
higher acrylamide concentration. It is also important
to note that the standard SDS PAGE may not give
an accurate measurement of Mr when samples are
not reduced or heated. Chymotrypsin consistently
migrated to Mr 36,000 when loaded unheated and
run on SDS PAGE, even though its correct Mr is
approximately 24,000. Unfortunately, few, if any,
proteases can be reactivated following electrophoresis if they have been first completely denatured.
TABLE 1
Sample
Elastase
Mr 22500a
Chymotrypsin
Mr 24,000
ScPrB
Mr n/a
Gel
%T
SDS
PAGE
Gelatin
SDSPAGE
Overestimate
(%)
7.5
10
12.5
7.5
10
12.5
7.5
10
12.5
ndb
21,300
24,500
35,500
36,300
33,800
56,200
57,500
57,500
nd
39,400
36,300
79,400
60,300
53,703
79,400
74,100
65,300
nd
84
48
123
66
58
41
29
14
a
Mr as determined from published amino acid sequences. The
amino acid sequence of ScPrB has not been determined.
b
nd, not determined.
REFERENCES
1. Heussen, C., and Dowdle, E. B. (1980) Anal. Biochem. 102, 196
202.
2. Garcia-Carreno, F. L., Dimes, L. E., and Haard, N. F. (1993)
Anal. Biochem. 214, 6569.
3. Harrington, D. J., and Russel, R. B. (1994) FEMS Microbiol.
Lett. 121, 237242.
4. Michaud, D., Faye, L., and Yelle, S. (1993) Electrophoresis 14,
9498.
5. North, M. J., Cotter, D. A., and Franek, K. J. (1990) J. Gen.
Microbiol. 136, 835840.
6. Rodier, M-H., El Moudni, B., Ghazali, M., Lacroix, C., and Jacquemin, J-L. (1994) Exp. Mycol. 18, 267270.
7. Gordon, L. J., and Lilly, W. W. (1995) Curr. Microbiol. 30, 337
343.
FIG. 1. Migration of standard protein markers (10-kDa ladder,
Gibco-BRL) in standard SDSPAGE (10% T) and gelatinSDS
PAGE (0.5% gelatin, 10% T).
/ m4846$$nat
12-21-95 19:54:44
abnt
AP-Anal Bio
142
ANALYTICAL BIOCHEMISTRY
/ m4846$$nat
12-21-95 19:54:44
abnt
AP-Anal Bio