GE SEMIPHOR TRANSPHOR Series Operating Instructions Manual page 59

Uneven band transfer
Problem
Uneven band transfer
Inefficient binding to membrane
Part
Chemical parameters
Membrane parameters
SEMIPHOR TRANSPHOR UNIT series Operating Instructions 29281777 AA
Solution
The blotting paper and membrane must be the
same size as the gel or 1 to 2 mm smaller. Larger
sizes will provide an electrical path for current to
bypass the gel solution.
Different proteins will transfer at different rates
depending on size and net charge.
Function
Prepare protein transfer buffer without SDS. (SDS
can improve transfer efficiency but can interfere
with protein binding to a PVDF membrane.)
Verify the optimal amount of methanol required
for the membrane type and check the buffer solu-
tion. Add 10% to 20% methanol to the transfer
buffer to enhance binding to nitrocellulose.
Wear gloves when handling membranes.
Store membranes at ambient temperature out of
direct sunlight to keep the membranes activated.
Check shelf life of nitrocellulose membrane, re-
place if necessary.
Use a membrane with a smaller pore size (0.10 to
0.20 µm) if proteins pass through the membrane,
or use a different membrane type.
Place a membrane both over and under the gel if
you suspect one protein is moving in the opposite
direction from the majority of the proteins. Check
both membranes for protein(s).
Check if too much sample is available for the
binding surface area by applying two membranes
instead of one. If "blow through" occurs, reduce
the sample load.
7 Troubleshooting
59