Bio-Rad Sequi-Blot™ PVDF Membrane - Manual (Page 5)

ous solutions. If the membrane becomes
dry prior to blotting, repeat steps 1 and 2 to
rewet it.
Section 3
Dot Blotting
Dot blotting requires special precautions to
insure that protein is bound to the PVDF mem-
brane before it dries. When the membrane is
dry, protein molecules will not bind tightly and
will be washed off in subsequent analysis steps.
For protein sequencing, the amount of protein
bound is critical. For this application it might be
better to try the direct adsorption method for
attachment of proteins.
3 Direct adsorption con-
sistently provides 90% binding of small
amounts of protein. In this method, up to
300 pmol of protein is suspended in 200 µl of
buffer and incubated with a small piece (5 x 9
mm) of wetted PVDF membrane for 1 hour
5
1. Immerse the membrane in 100% methanol
for a few seconds, until the entire mem-
brane is translucent. In methanol, it wets
immediately. (Solutions containing 50%
methanol concentration can be used to pre-
wet the membrane.)
2. Transfer the wetted membrane to a vessel
containing transfer buffer or water.
Incubate in buffer until it is equilibrated
(² 2-3 minutes). The membrane will float
on the surface of the buffer until complete-
ly equilibrated. After it is equilibrated it can
be easily submerged into the aqueous solu-
tion. At this point, the membrane is ready
to bind proteins in any blotting application.
3. After the membrane has been wetted with
buffer, do not allow it to dry (white spots
will form where the membrane is dry).
Protein will not bind to the dried mem-
brane, and dry spots will not rewet in aque-
4
LIT240c.qxd 6/23/98 11:42 AM Page 4