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

Mini-Trans-Blot Electrophoretic Transfer Cell 23
5. Charge-to-mass ratio is incorrect.
• Try a more basic or acidic transfer buffer to
increase protein mobility.
6. Protein is precipitating in the gel.
• Try using SDS in the transfer buffer. SDS can
increase transfer efficiency, but can also reduce
binding efficiency to nitrocellulose and affect
reactivity of some proteins with antibodies
• An excess of methanol will lead to protein
precipitation. Try decreasing methanol content
7. Methanol in the transfer buffer is restricting elution.
• Reduction of methanol results in increased
transfer efficiency of proteins from the gel, but it
also diminishes binding to nitrocellulose
8. Gel percentage too high.
• Reduce %T (total monomer) or %C (crosslinker).
A 5.26% C (with bis as the crosslinker) will
produce the smallest pore size gel. Decreasing
from this concentration will increase the pore size
and increase transfer efficiency
Poor transfer—nucleic acid
1. Gel percentage is too high.
• Reduce the %T or %C in the acrylamide gel or
reduce % agarose in an agarose gel
• Prior to transfer, cleave DNA in 0.25 M HCl or
RNA in dilute NaOH
2. Transfer time is too short or power conditions are too
low.
• Increase the transfer time, or try high intensity
transfer
3. DNA or RNA cannot be transferred electrophoretically
to nitrocellulose, since high salt concentrations are
required for efficient binding.
• Use Zeta-Probe membrane instead of
nitrocellulose