Hoefer TE42 - Manual (Page 24)

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p16
problem solution
Diffuse band patterns Transfer immediately after electrophoretic separation. If equilibrat-
ing before the transfer, shorten or eliminate the equilibration time or
move the gel to the cold room during equilibration.
If transfer buffer contains methanol (≥10%), equilibrate the gel in
transfer buffer for 30 minutes to allow it to shrink before assembling
the stack. Note: Because methanol causes the gel to shrink slightly,
large molecules may migrate more slowly.
Take care that the gel is held firmly against the membrane and that it
does not shift once contact is made.
If excess heating occurs during the transfer, lower the temperature of
the cooling fluid in the heat exchanger.
Check that the preferred binding surface of the membrane (if any)
contacts the gel.
Inefficient binding to membrane
Chemical parameters
Fix or crosslink the molecule onto the membrane according to the
requirements of the nucleic acid, protein, or membrane type.
Prepare protein transfer buffer without SDS.
Verify the optimal amount of methanol required for the membrane
type and check the buffer solution. Add 10–20% methanol to the
transfer buffer to enhance binding to nitrocellulose.
Membrane parameters Wear gloves when handling membranes.
Store membranes at ambient temperature out of direct sunlight to
keep the membranes activated.
Use a membrane with a smaller pore size (0.10–0.20 µm) if proteins
pass through the membrane, or use a different membrane type.
Place a membrane both over and under the gel if you suspect one
protein is moving in the opposite direction from the majority of the
proteins. Check both membranes for protein(s).
Check if too much sample is available for the binding surface area
by applying two membranes instead of one. If “blow through” occurs,
reduce the sample load.
For more troubleshooting hints,
refer to Bjerrum, O.J. et al. (1988).