Poor Protein Transfer (Western Blot (Sigma Protocol))

Question

Accepted Answer

Proteins do not transfer efficiently from gel to membrane, resulting in weak or no signal Common causes: 1) Membrane did not wet uniformly before transfer 2) Transfer time inappropriate for protein size 3) High methanol concentration in transfer buffer preventing protein release 4) Air bubbles trapped between gel and membrane Solutions: 1) Check transfer by staining membrane with Ponceau S prior to blocking 2) Optimize transfer time: small proteins (<10 kDa) may transfer through membrane, larger proteins may require longer times 3) Reduce methanol concentration in transfer buffer or add 0.005-0.01% SDS to enhance transfer 4) Use blot roller to carefully remove air bubbles between layers during stack assembly