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