Low Molecular Weight Protein Signal Loss

Symptom

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.

1. 1 Over-transfer or 'blow through': small proteins pass completely through membrane pores during extended transfer
2. 2 Standard 0.45 µm pore size nitrocellulose membrane allows low MW proteins to pass through
3. 3 Transfer time optimized for high MW proteins too long for low MW targets

1. 1 Use nitrocellulose transfer membrane with 0.2 µm pore size to minimize loss of proteins less than 25-30 kDa
2. 2 Reduce transfer time for low MW proteins: use 1-1.5 hours instead of standard 2 hours at 70V (200-250 mA)
3. 3 Monitor transfer efficiency by Ponceau S staining immediately after transfer to verify protein retention
4. 4 Avoid over-transfer by checking membrane and gel after transfer: protein should be on membrane, not remaining in gel

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