Bio-Rad Trans-Blot® Plus Cell - Manual (Page 28)

Swirls or missing bands; diffuse transfers
1. Poor contact between the membrane and the gel. Air bubbles or excess buffer
remain between the blot and gel.
• Use the roller carefully to roll over the membrane in both directions until
air bubbles or excess buffer is removed from between gel and membrane,
and complete contact is established.
• Use thicker filter paper in the gel/membrane sandwich.
• Replace the fiber pads. Pads will compress and degrade with time, and
will not hold the membrane to the gel.
2. The membrane is not properly wet or has dried out.
• White spots on nitrocellulose membrane indicate dry areas where
protein will not bind. If wetting does not occur immediately by immersion
of the sheet in transfer buffer, heat distilled water until just under the
boiling point, and soak the membrane until completely wet. Equilibrate in
transfer buffer until ready for use.
• Because of the hydrophobic nature of PVDF, the membrane must be
prewet in methanol prior to equilibration in aqueous transfer buffer. Follow
the directions in the product insert.
3. The gel electrophoresis may be at fault.
• Artifacts of electrophoresis may occur as a result of poor gel polymerization,
inappropriate running conditions, contaminated buffers, sample overload,
etc. Consult your electrophoresis manual for more details.
Gel cassette pattern transferred to blot
1. Contaminated or thin fiber pads are used.
• Replace the fiber pads, or thoroughly clean the contaminated pads.
2. The transfer buffer is contaminated.
• Make fresh solutions.
Poor binding to the membrane — Nitrocellulose
1. 20% methanol in the transfer buffer is generally optimal for protein binding.
• Make sure the buffer contains the proper amount of methanol.
2. Proteins may be transferring through the nitrocellulose.
• Use PVDF or 0.2µm nitrocellulose (smaller pore size). Decrease the voltage
if using the high intensity option.
• Place an additional piece of nitrocellulose membrane in the gel sandwich
and analyze this added piece for evidence of proteins that may have
transferred completely through the first piece.
3. Proteins <15,000 Da may show diminished binding to 0.45 µm nitrocellulose, or
may be washed from the membrane during assays.
• Use PVDF or nylon membrane, which have higher binding capacities.
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