Thermo Scientific OWL HEP-1, Руководство по эксплуатации и техническому обслуживанию

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4-2 Semidry Electroblotter Thermo Scientific
Section 4
Troubleshooting
Problem Cause Solution
Nitrocellulose membranes
Insufficient binding of
proteins to the mem-
brane
• Over-transfer through the
membrane
• Use 0.2micron pore size nitrocellulose instead
of 0.45micron, or use PVOF with a higher binding
capacity.
• Not enough methanol in the
transfer buffer
• Nitrocellulose binds proteins best when 20%
methanol is used in the transfer buffer.
• Low MW proteins are not
binding well or are being washed
away
• Use glutaraldehyde to crosslink the proteins to
the membrane and use Tween-20 in the wash
steps.
• SOS is preventing binding • Eliminate SOS in the transfer buffer.
PVDF
Smeared or swirled
transfer and missing
bands
• Membrane was dried out before
it was added to the transfer sand-
wich
• Membrane should be completely gray and
slightly translucent when added to the sandwich.
If it has dried out, rewet in methanol and equili-
brate in transfer buffer.
• Alcohol was not used to prewet
the membrane
• PVDF is hydrophobic and requires a short soak
in methanol prior to transfer.
• Air spaces are interfering with
contact between the gel and the
membrane
• Roll a test tube or pipet over the membrane
(make sure it is clean) before putting the rest of
the filter paper on the sandwich. Transfer will not
occur where the gel is not in contact with the
membrane.
• Electrophoretic conditions were
incorrect or not ideal
• Running conditions, sample preparation, per-
centage acrylamide, and many other variables
can affect the migration and resolution of pro-
teins. Review your electrophoresis conditions.
• Transferring at too high a current • Refer to the Running Conditions table.
• Membrane was not thoroughly
wetted.
Always pre-wet the membrane according to the
manufacturer’s instructions. White spots indicate
the dry areas of the membrane.
• Too much current
• Running at constant voltage can cause power
fluctuations that will cause overheating. A buffer
that has not been made correctly or that has too
high in ionic strength can also burn a gel by over-
heating. A cracked and dry gel often is an indica-
tor of overheating.