Hoefer TE70X - Manual (Page 22)

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4. Troubleshooting
problem solution
Incomplete transfer
Blank or faint areas on
Remove trapped air pockets between the gel and
the membrane membrane during stack assembly.
Use buffer with a lower ionic strength.
Molecules do not migrate Check all electrical connections. Confirm that current
out of gel is flowing through the transfer stack.
Check that the buffer pH is close to the intended pH.
Most buffers should not be titrated. Make fresh buffer.
Use 3.5 mM 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.
Increase the 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.
If using a non-nitrocellulose membrane, avoid includ-
ing 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 using a
transfer buffer with a different pH. Lower pH (< 6 – 7)
increases the positive charge on proteins; higher pH
( > 6 – 7) increases the negative charge on proteins.