Bio-Rad Criterion™ Blotter - Manual (Page 17)

2. The membrane is not properly wet or has dried out.
• White spots on the 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 100%
MeOH 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 be produced by poor 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 worn 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 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 nylon (higher binding capacities) or 0.2 µm nitrocellulose (smaller
pore size). Decrease the voltage.
3. Proteins <15,000 daltons 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.
• Use only Tween-20 detergent in the wash and antibody incubation steps. Reduce or
eliminate the more stringent washing conditions.
4. SDS in the transfer buffer will reduce binding efficiency of proteins.
• Reduce or eliminate the SDS from the transfer buffer.
5. The membrane may not be completely wet.
• White spots on the 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.
Poor Binding to the Membrane—PVDF
1. The membrane may not be completely wet.
• Because of the hydrophobic nature of PVDF, the membrane must be prewet in
100% MeOH prior to equilibration in aqueous transfer buffer. Follow the directions
in the product insert.
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