Bio-Rad Mini Trans-Blot® Cell - Manual (Page 31)

Mini-Trans-Blot Electrophoretic Transfer Cell 25
4. The gel electrophoresis may be at fault.
• Artifacts of electrophoresis may be produced
by poor polymerization, inappropriate running
conditions, contaminated buffers, sample
overload, etc
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 without
binding, and recirculate through the tank blotting
system.
• Reduce the amount of protein on the gel, and
SDS in the transfer buffer. Reduce transfer
duration or add a second sheet of membrane to
bind excess protein
3. The transfer buffer is contaminated.
• Make fresh solutions. Transfer buffer solution
cannot be reused
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 (higher binding capacities) or 0.2 μm
nitrocellulose (smaller pore size). Decrease the
voltage if using the high intensity option
3. Mixed ester celluloses bind proteins poorly.
• Use pure nitrocellulose