Bio-Rad Trans-Blot® Plus Cell - Manual (Page 29)

• Use 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 it is 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
completely soaked in methanol 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.
3. Proteins may be transferring through the membrane.
• Decrease the voltage if transferring under high intensity conditions.
• Place an additional piece of PVDF membrane in the gel sandwich and
analyze this added piece for evidence of proteins that may have
transferred completely through the first piece.
4. SDS in the transfer buffer will reduce binding efficiency of proteins.
• Reduce or eliminate the SDS from the transfer buffer.
Power conditions are too high
• Always check the current at the beginning of the run. The current may be too
high for a particular voltage setting. If the buffer is prepared improperly, the
conductivity may be too high, resulting in excessive power delivered to the cell.
See the power guidelines for specific applications in Section 3.
Immune-Specific Detection
Overall high background, low signal, or lack of development of positive control.
• Consult instructions for immune detection kit or reagents.
Total Protein Detection
Consult stain or detection kit user manual.
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