pGEX/GST gene fusion problems-> HELP!

[Mark Evans]

I have used this system extensively in the past. From what I remember, 
PMSF activity is pH dependant and it's life span is very short (about30 
min). I would recommend using a cocktail of several different inhibitors 
to different classes of proteases, including cysteine proteases. Also 
make sure to add them to your initial lysis buffer and carry them 
throughout the purification process. Standard practice is to add fresh 
inhibitors whenever you change buffers. Good Luck