What parameters should be consider when transferring HMW proteins?

Proteins of high molecular weight (HMW) require specific transfer conditions, different to those for proteins of low molecular weight (LMW).

For HMW proteins, the following conditions are recommended:
  • Membrane with larger pore size (0.45 µm)
  • Methanol in a transfer buffer should be decreased to 10%
  • SDS concentration should be increased
  • Wet (tank) transfer is recommended
  • Transfer time may need to be extended, typically it is 1-2 hours at 100 V or overnight at 30V (for wet transfer)

To check that the protein of interest is transferred correctly, stain the gel after the transfer with Coomasie R250. If there are bands in the region of MW of your protein of interest, the protein transfer conditions have to be modified. A stained gel should not show any bands after transfer.
 


  
Comparison of conditions to transfer HMW and LMW proteins


Agrisera detailed Western blot protocol 

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