Low Molecular Weight Protein Signal Loss (Western Blot (CST Guide))

Question

Accepted Answer

Proteins smaller than 25-30 kDa show weak or absent signal despite adequate expression levels. Higher molecular weight proteins from the same sample transfer successfully. Common causes: 1) Over-transfer or 'blow through': small proteins pass completely through membrane pores during extended transfer 2) Standard 0.45 µm pore size nitrocellulose membrane allows low MW proteins to pass through 3) Transfer time optimized for high MW proteins too long for low MW targets Solutions: 1) Use nitrocellulose transfer membrane with 0.2 µm pore size to minimize loss of proteins less than 25-30 kDa 2) Reduce transfer time for low MW proteins: use 1-1.5 hours instead of standard 2 hours at 70V (200-250 mA) 3) Monitor transfer efficiency by Ponceau S staining immediately after transfer to verify protein retention 4) Avoid over-transfer by checking membrane and gel after transfer: protein should be on membrane, not remaining in gel