Bio-Rad Trans-Blot® Cell - Manual (Page 20)

4. The gel electrophoresis may be at fault.
• Artifacts of electrophoresis may be produced by poor polymerization,
inappropriate running conditions, contaminated buffers, sample over-
load, 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. Excessive amounts of protein were loaded on the gel, or too much SDS was
used in the transfer buffer. Proteins can pass through the membrane with-
out binding, and recirculate through the tank blotting system.
• Reduce the amount of protein on the gel, and SDS in the transfer buffer.
Add a second sheet of membrane to bind excess protein.
3. The transfer buffer is contaminated.
• Make fresh solutions.
Poor binding to the membrane — Nitrocellulose
1. Nitrocellulose requires 20% methanol in the transfer buffer for optimal
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 or move the electrodes to the
standard position if using the high intensity option.
3. Mixed ester celluloses bind proteins poorly.
• Use pure nitrocellulose.
4. Proteins <15,000 daltons may show diminished binding to 0.45 µm nitro-
cellulose, or may be washed from the membrane during assays.
• To increase stability of binding, proteins can be crosslinked to
nitrocellulose with glutaraldehyde.
• Use PVDF or nylon membrane, which have higher binding capacities.
• Use Tween-20 detergent in the wash and antibody incubation steps.
Reduce or eliminate the more stringent washing conditions.
5. SDS in the transfer buffer will reduce binding efficiency of proteins.
• Reduce or eliminate the SDS from the transfer buffer.
6. 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 alcohol prior to equilibration in aqueous transfer buffer.
Follow the directions in the product insert.
2. The membrane may have been allowed to dry during handling.
• A completely wet membrane has a gray, translucent appearance. White
spots will form on the surface of the membrane, indicating that it has
been allowed to dry. Since proteins will not bind to the dry spots, rewet
the membrane with methanol and re-equilibrate in transfer buffer.
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