GST tagged protein purification - (Oct/16/2008 )

Hi,
I have GST tagged protein which is cloned in the BL21. When I checked the expression with SDS page and western blot I had the band. I decreased the tempreture to 25 and checked different time and concentrations and found that between 1 to 3hrs in 25 D my protein is soluble. Then start to purify it. But at the end I dont have any OD with bradford assay. And this purification is know an issue for me. I dont know why its not purifying. I am using GST tagged purification module kit fromGE health care.

Do you have any suggestion.

Thanks,
Sara

Hi P,
The method that I am using is through the column. But because at this stage I am doing small scale I just wash the slurry 3 times with PBS, then I add my protein and incubate it at cold rom with gentle shake for 2 hrs. then Centrifuge for 5 min and remove the supernatant, them 3 times wash and incubate with elution buffer for 10 min in frige and again 5min centrifuge and check the result with SDS page.
The elution buffer that I use is reduced glutation which comes with the kit and I checked the PH today. It is around 8. As you recomend.
I was thinking may be I over sonicate my sample.
can you give me any sugestion for sonication?
I read some where 3x 10 sec but the didnt mention any amplitude? do you think 50% is good or I should use the less amount.

Cheers,
Sara

Hi,
Sonication time depends on sample size, 3 X 10 sec is ok for small samples (i.e. pellet from a 50 ml culture?) so I don't think you would have over sonicated - maybe even under sonicated! Did you check the pellet after sonication to see if your protein is there (i.e. not released from the cells) or in the supernatant?

50% amplitude is the standard condition, I've never tried increasing or decreasing it.

P

Hi,
Che cked the palate and eluted samples and purified one as well. Halph the protein is in the pallate. the rest in the elute. No thing in the purified one. Actually I am dealling with two problem:
1- inclusion bodies.
2- not gething the purified protein.

For the first part. I am decreasing the temp to 20, and I am checking the IPTG concentration of 0.1, 0.4 and 0.7. ( I am doing it today. My sample size is 3 ml. and I am inducing them for 3 hrs.

For the second problem probably I should check the sonication! The timing that I mentioned is what I want to do. Last time my Amp was 90% and I did it for 4 x 50sec.

3- I have one other problem too. when I am doing the small amount I dont get very much pallet and so no inclusion body problem. but when I increase the volume I get more pallet and so inclusion as well!

Do you think is it possible after sonication and separation of the super natant, if I add the same amount lysis bufer and sonicate and centrifuge and use it for purification it is going to work?


Thanks,
Sara

If you're concerned about the sonication step, have you tried freeze-thawing with lysozyme and DNase? 3x in liquid N2, thawing in a beaker of water. Don't forget the protease inhibitors! Very gentle technique, but slightly longer than sonication. Then do a hard spin, and run S/N and pelleted material on a gel (resuspend the pellet in the same volume as the S/N so you get relative amounts in each fraction).

As for the rest of your protocol, you say you purify on the column, but then you describe a batch-mode purification. For what it's worth, I think batch is better for these small-volume experiments. It is possible to scale-up and load the beads into an empty column body, rather than try to re-optimise for a pre-packed column system.

HI,
I have tried the freeze and thaw method but never added DNase or RNase. Do you know Which DNase and RNase I should add?

Thanks,
Sara

Hi,
Thanks for your reply. yesterday I did sonication with very mild situation 5 min, 10s/40s rest. With DNAse 1 .My protein samle didnt get transluent. Do you always , when lysed the bacteria for purification , get the transluent sample before centrifuge? How long you think the sonication can take? whith this situation.

Sara