Help for a purificaltion newbie - (Oct/05/2010 )
Hello all!
I have started in a lab where we have no experience purifying protein, but it is something that I need to do. I have found a protocol by looking online, but was wondering if anyone might have some advice for me. I am using the pGEX-4T vector series, and would like to purify my protein both with and without the GST tag in the end. From what I have gathered, I can use thrombin to cleave off GST, and elute with reduced glutathione to keep the GST tag, does this sound correct?
Any advice or protocols you could share with me would be most appreciated.
jdklove18 on Tue Oct 5 17:58:49 2010 said:
Hello all!
I have started in a lab where we have no experience purifying protein, but it is something that I need to do. I have found a protocol by looking online, but was wondering if anyone might have some advice for me. I am using the pGEX-4T vector series, and would like to purify my protein both with and without the GST tag in the end. From what I have gathered, I can use thrombin to cleave off GST, and elute with reduced glutathione to keep the GST tag, does this sound correct?
Any advice or protocols you could share with me would be most appreciated.
Hola, If you want to do the digestion in the colum, you have to stop flow, let some hours and then elute your digested protein with buffer without glutation. Other way would be elute the protein and digest the amount that you want; repeat the colum separating digested protein eluted in the flowthrought, and the GST bound to the resin.Probably in the GE healthcare page you could download a suitable method. Good Luck
Hi! My personal experience with GST: if you are a newbie, try eluting the protein, digesting and repeating the purification as he said... the "in column digest" never worked for me!
protolder on Wed Oct 6 05:33:55 2010 said:
jdklove18 on Tue Oct 5 17:58:49 2010 said:
Hello all!
I have started in a lab where we have no experience purifying protein, but it is something that I need to do. I have found a protocol by looking online, but was wondering if anyone might have some advice for me. I am using the pGEX-4T vector series, and would like to purify my protein both with and without the GST tag in the end. From what I have gathered, I can use thrombin to cleave off GST, and elute with reduced glutathione to keep the GST tag, does this sound correct?
Any advice or protocols you could share with me would be most appreciated.
Hola, If you want to do the digestion in the colum, you have to stop flow, let some hours and then elute your digested protein with buffer without glutation. Other way would be elute the protein and digest the amount that you want; repeat the colum separating digested protein eluted in the flowthrought, and the GST bound to the resin.Probably in the GE healthcare page you could download a suitable method. Good Luck