Hi everyone. I hope you guys can help me troubleshoot my problem.
I cloned my insert into pMAL c5x expression system and transformed it into NEB express E. coli. About a year ago I was able to express the protein in the soluble fraction. But recently I needed more protein so I started protein purification again. However, not only is my protein insoluble but almost all the protein is insoluble. I followed the same procedure I did a year ago ( 13 hour overnight culture; Induce at OD = 0.5; induce for 4 hours with 4mM IPTG). So far some trouble shooting tactics include. Making fresh AMP stock,IPTG stock, induce at lower temperature, lower IPTG concentration, and using a lysis buffer instead of the recommended column buffer (in-case the salt concentration was wrong).
Do you guys have any suggestion as to how I should troubleshoot this problem? Any help will be well appreciated.
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#2
Adrian K
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Posted 05 May 2011 - 08:05 PM
During your overnight culture, do you grow in LB with glucose? (to avoid leaky expression)
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..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
#3
toshuff
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Posted 05 May 2011 - 10:59 PM
adrian kohsf, on 05 May 2011 - 08:05 PM, said:
During your overnight culture, do you grow in LB with glucose? (to avoid leaky expression)
Yep 2g of glucose per liter. Thanks for the reply. I think I'm going to try to transform the vector into another cell line.
#4
protolder
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Posted 05 May 2011 - 11:40 PM
toshuff, on 05 May 2011 - 10:59 PM, said:
adrian kohsf, on 05 May 2011 - 08:05 PM, said:
During your overnight culture, do you grow in LB with glucose? (to avoid leaky expression)
Yep 2g of glucose per liter. Thanks for the reply. I think I'm going to try to transform the vector into another cell line.
