Thrombin digestion for GST - ppt problem (Jun/01/2008 )
Dear all, I am trying to purify GST-tagged 54kD protein. the problem is as follow: after elution into 50mM Tris buffer (using 600mM glutathione reduced) into 1ml, I buffer exchange into PBS 7.3ph and conc. protien to 300ul(giving approx 5mg of protien). Then i added about 50Units of PBS dissolved Thrombin but I soon as I do so..protein gets ppt. I can see ppt immedialtely as I do add thrombin solution. Any idea ...
I don't recall my experience when I had done it in the last century, but see if you find any tips here:
5mg of protein in 300ul is very concentrated! Its likely that when you cleave off the GST tag your protein becomes free to oligomerise to such an extent that it crashes out of solution. I would dilute the protein before doing the cleavage and add in detergents like up to 0.5 mM Trition X-100 or 0.1% NP-40 to reduce olgomerisation. Do you really need your protein so concentrated?
Thanks for ur reply but I resolved issue by adding BME.
Thanks for nice site VADLO.