Amersham Biosciences Hoefer DALT, User Manual, Page: 63 / 70

Hoefer DALT System Troubleshooting
Amersham Biosciences
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Diffuse band patterns
• Transfer immediately after electrophoretic separation. If equilibrating 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 before in
transfer buffer for 30 minutes to allow it to shrink before assembling the
stack. Because methanol causes the gel to shrink slightly, large molecules
may migrate more slowly.
• Make sure 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 setting
of the circulating cooler.
• Check that the preferred binding surface of the membrane, if any, contacts
the gel.
Inefficient binding to membrane
Chemical parameters
• 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
µ
m) if proteins pass
through the membrane, or use a different membrane type.
• If you suspect one protein is moving in the opposite direction from the
majority of the proteins, place a membrane both over and under the gel.
Check both membranes for proteins.
• 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.