Solution
Problem
Check all electrical connections. Confirm that
current is flowing through the transfer stack.
Molecules do not migrate out of
gel
Increase transfer period. Large fragments may
require an additional hour.
Do not use staining or fixing agents on the gel
before transfer.
Use a thinner gel.
Reduce the gel acrylamide concentration.
Check that the buffer pH is close to the intended
pH. Most buffers should not be titrated. Make fresh
buffer.
Use 3.5 mM sodium dodecyl sulfate (SDS, 0.1%) in
the transfer buffer.
Add several more sheets of buffer-saturated
blotting paper to each side of the gel sandwich
so that more buffer is present during the transfer.
If using a non-nitrocellulose membrane, avoid in-
cluding methanol in the transfer buffer or reduce
the amount to the minimum possible.
Use reagent-grade chemicals.
Increase the net charge on the protein by chang-
ing to a transfer buffer with a different pH. Lower
pH (< 6 to 7) increases the positive charge on
proteins; higher pH (> 6 to 7) increases the nega-
tive charge on proteins.
SEMIPHOR TRANSPHOR UNIT series Operating Instructions 29281777 AA
57
7 Troubleshooting
