I have cloned a gene with N terminally fused GST in pGEX6P1. II expressed it in BL21(DE3) pLysS cells induced at 0.6 OD 600 with 1mM IPTG, 16 degree celcius overnight in 4 ml of 2YT medium supplied with AMP+Chloramphenicol+0.1% glucose. Then I prepared the Whole Cell Extract from 1 ml of culture from each sample, pelleted and boiled with 50 micro liter of 5X SDS loading dye, followed by 13,200 rpm spin for 10 minutes at 4 degree celsius and 25 microliters of supernatant from each sample was taken and loaded on 12% SDS-PAGE.
loading order as follows
pGEX6P1 Uninduced colony 1
pGEX6P1 induced colony 1-----------------GST expressed 26 kDa
pGEX6P1 Uninduced colony 2
pGEX6P1 induced colony 2 ---------------GST expressed 26 kDa
pGEX6P1-with gene, Uninduced colony 1
pGEX6P1 with gene, induced colony 1-------------GST expressed and GST fusion protein expressed (2 bands) 26kDa and 110 kDa respectively.
pGEX6P1 with gene, uninduced colony 2
pGEX6P1 with gene, induced colony 2------ only GST fusion protein expressed (single bands) 110 kDa.
Can anyone explain why the GST was not expressed in pGEX6P1 with gene colony 2 induced? In this lane I could only observe expression of fusion protein of 110 kDa but not GST.
Hi-
I'm not seeing the gel, but when you observe the GST after induction it generally comes from proteolysis, you should normally see only one band at 110 Kd (colony 2). However this is classical with GST tag to observe some proteolysis and thus a GST band around 26Kd after induction but don't worry for your clone 2, this is the right one!
Best,
Hey, is the level of protein expressed by colony 2 lesser? There might be gst present but may be just too faint to be seen!
hi Simon Lebaron · thank you for your suggestion
i have attached the file for you reference,
please tell me the actual problem.
Hi,
so usually we streak through the plate of expression clones and do not test individual ones for expression anymore. By streaking through to innoculate we make sure to get a rather average expression then a specifically high expressing clone. So we loose, but we also win.
In your case you are there, right? Yout got it. Take the clone and put the supernatant on a affinity column and wou will get your protein. If you want to get rid of the extra GST, perform on column digest with your protease (if you have a recognition site between protein of interest and GST) or elute it and run gel filtrations and ion exchanges. should be fine.
cheers Sven
thank you Mr Seven for your kind suggestion i will going to try it
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