Bio-Rad Mini Trans-Blot® Cell - Manual (Page 32)

26 Mini-Trans-Blot Electrophoretic Transfer Cell
4. Proteins <15,000 daltons may show diminished
binding to 0.45 μm nitrocellulose, or may be washed
from the membrane during assays.
• To increase stability of binding, proteins can be
crosslinked to nitrocellulose with glutaraldehyde
• Use PVDF membrane, which has 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