Hoefer TE42 - Manual (Page 28)

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p20
Protein transfers
Study summaries
Gershoni and Palade (1982) investigated factors
affecting protein recovery from SDS gels to
nitrocellulose or DBM paper. According to their
findings, methanol in the Towbin buffer system
is necessary to achieve efficient binding to nitro-
cellulose. Methanol improves binding in part by
removing protein-bound SDS. In the absence of
methanol, labeled bovine serum albumin (BSA)
passes through at least five layers of membranes.
Methanol may cause a gel to shrink, however,
so the elution rate decreases. By using a cationic
membrane (such as nylon), which binds the
proteins more efficiently, and omitting methanol
from the transfer buffer, Gershoni and Palade
obtained a much more quantitative transfer.
The disadvantage of cationic membrane is that
protein stains also bind well, so that the stain-
ing background tends to be very high. Properly
quenched, however, this paper can be used for
antibody detection or other overlay methods of
protein identification. A summary of membrane
type and recommended methanol concentration
follows:
membrane type methanol %
Charged nylon 0
Nitrocellulose ≤ 20
PVDF ≤ 15
Some workers have reported to us that a low
concentration of SDS (0.1%) improves the trans-
fer of protein from an SDS gel. Burnette (1981)
and Symington et al. (1981) investigated the
effect of the molecular weight of protein. Gibson
(1981) describes a method to increase the extent
of transfer of large proteins by limited cleavage
with pronase during transfer.