However, when I do my purification and elute the proteins off the beads, I see two significant degradation bands around 25 kD and I observe co-purification with my protein significant quantities (more than my protein) of DnaK protein, which is a Hsp70 chaperon (this is seen in the gel below as the huge band above my protein band, which is marked with an arrow) In my lysis buffer, I have 1mM PMSF + Roche protease inhibitor tablet and I do all purification steps in the cold room at 4 C. Hence, I cannot think of any reason why my protein would be degraded in such large quantities unless my lysis condition is too harsh. I have the evidence of this harsh lysis condition because the co-purification of DnaK, which according to GE lifesciences (company that made the GST beads) is due to harsh lysis conditions. This got me thinking, is it possible for soniciation to lyse my cells to be too harsh? I soniciate for 3 times @ 10 secs each with 30 secs rest in between. I repeat this for a total of 7 times which means I essentially soniciate 21 times for 10 secs each time. I keep my all my samples on ice during soniciation and cool the probe whenever necessary.
Edited by p3t3r1, 15 August 2009 - 02:30 PM.