|The most common mistake when working with proteins of low molecular weight is that the applied protein transfer conditions are not adjusted properly. This results in a lack of signal in Western blot. |
Low molecular weight proteins (LMW) migrate much easier from the gel through the membrane, and conditions that have always worked for transfering other porteins, may not work anymore.
For LMW proteins:
- Membrane pore size has to be smaller.
-Methanol content in the transfer bufffer has to be increased, while SDS content decreased.
- Semi-dry protein transfer seems to work better.
To check if a LMW target protein went through the membrane during protein transfer, one can place one more membrane in the sandwich and stain both membranes with Ponceau S reversible stain. If the first membrane is blank below 20 kDa, while the second membrane stains, it means that the voltage needs to be changed, and conditions of the protein transfer need to be adjusted.
Both membranes should be separated by Whatman sheets when assembling the sandwich.