Severe protein degradation (Protein Extraction)

Question

Accepted Answer

SDS-PAGE shows obvious low-MW degradation bands and a smear/tail. Target band is faint or absent on Western. Common causes: 1) Endogenous protease activity not inhibited 2) Operation took too long or temperature too high 3) Repeated freeze-thaw or vigorous vortexing 4) Protease inhibitor missing or already lost activity 5) Sample stored under wrong conditions Solutions: 1) Work on ice throughout; shorten processing time 2) Add fresh protease inhibitor cocktail (e.g. cOmplete) to the lysis buffer right before use 3) Avoid repeated freeze-thaw; aliquot samples 4) Use lysed sample immediately or store at −80 °C 5) Avoid violent vortexing and harsh agitation