Pharmacia Biotech Multiphor II User Manual page 65

Symptom
Trouble shooting guide to electrophoretic transfer
Symptom
Incomplete transfer
Poor transfer
Inefficient transfer
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Cause
Unevenly wetted electrode strips
Electrode strips too short
Cause
Gel concentration too high
Methanol present in transfer buffer
Transfer time too short
Field strength too low
Too low charge/mass ratio
Air trapped between gel and membrane
Too low binding efficiency
(molecules migrate from the gel,
but pattern is faint)
Field strength too high
(e.g. low Mol. Wt. DNA)
Transfer time too long
Pore size too large
8. Trouble shooting
Remedy
Electrode strips must be evenly
wetted and be neither too wet
nor too dry
The strips should be cut just
short of the edges of the gel
Remedy
Use reversible cross-linkers (e.g.
DATD, BAC, DHEBA) and
depolymerize gel before transfer.
Lower monomer concentration.
Convert molecule to smaller form
by limited digestion with
proteases (for proteins) or with
nucleases or acid hydrolysis (for
nucleic acids)
Remove methanol from transfer
buffer
Increase transfer time
Increase field strength
Change transfer buffer pH
further away from molecules pI.
Add 0.1% SDS
Carefully push out all air bubbles
from the layers of the transfer
sandwich
Use different immobilizing
membrane (DEAE, NC, DBM,
DPT). Immobilizing membrane
needs to be activated. Remove
interfering substances
(denaturants, detergents).
Raise/lower salt concentration.
Raise/lower pH
Lower field strength
Shorten transfer time
With nitrocellulose, use smaller
pore filters (0.1 or 0.7 µm)
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