Contamination in GST fusion protein purification - (Feb/09/2010 )

Hello all,
I am facing problem with GST fusion protein purification. I always get a lot of contaminating proteins during my protein prep. I use 1x TBS with 0.1 % Triton-X 100 and 10mM DTT. I give 4 * 10 CV washes after binding, but contaminnation is persistent. I pure proteins which shows only a single band in silver staining. Right now im doing in small batches i want to scale up my purification. I have also tried reducing my resin volume. I need some sugesstion on this!!!

I have one more query can i store my Glutathione elution buffer @ -20 coz it takes atleast 10 ml to adjust pH to 8. I need ony 2 ml of it. rest goes waste.


Regards

venkat

Hi Venkat,
You may try increasing NaCl concentration in your buffer, while you are doing washes.
You should run all the washes on SDS-PAGE. When you do elution, you should increase the GST in gradient fashion, may you can start with 1 mM, followed 5 mM, and finally 10 mM or whatever max. conc you are going upto. You can analyze all these on SDS-PAGE and find in which fraction your protein goes..!!

May be if you could put here the step wise procedure in points that you do, it will be easier for people to comment on specific steps, where they think you can improve.

Also, in silver staining even the slightest conc of contaminating protein will show a band, it is very sensitive close to 1ng (I think so). You should go for CBB and see how many bands you can see in the elute..

Hope this help.

Best regards,
Kaushik

Yes you can store Glut elution buffer at -20 degrees, in aliquots of 1 ml...no issues