First run uninduced, induced, protein before passing through the column, protein after passing through the column, wash, elutes if you have any on a single SDS PAGE gel and track where you lost your protein then accordingly change your parameters to purify your protein. Good luck.

Dear Dr. Mopidevi,

During my SDS PAGE, I always put between before and after induction sample and crude extract. My protein can be easily tracked because of its thick band. After purification I can easily track also my protein, but the quantity of the peak was much less, or even, looks be degraded by `something` compared to its after induction. Thank you very much for your suggestion

Dear Mr. Sharma,

Thank you very much for your suggestion to use triton X 100 and to get more information in Sambrook, I will try. According your prediction, Unfortunately, my protein is not in pellete.

i have no doubt, you have pretty much good amount of recombinant protein in the soluble form. Finally to recover high amount of protein, first it should bind to your Ni column. Here my question is how much protein bind to the column? This you can only verify by comparing the protein before passing through the Ni column, protein after passing through the Ni column on SDS-PAGE. Once you know that you have enough protein bound to the column then you can try to get maximum protein yield if not you need to consider recharging the Ni column.

Hallo Hasan

When you found your protein soluble, have you streak on a plate your positive E.coli culture (with the right antibiotics) before to inoculate a big culture? As antibiotics do not kill the E.coli but only stop their growth, if you prepare a stock glycerol from the first colony took from the plate after transformation, happens that you have in your culture also bacteria without plasmid. As AMP is inactivated after few h at 37°C the E.coli without plasmid overgrowth and your protein yield decrease. I suggest to go back at the small scale culture and check if you lose your plasmid during the big growth culture. In general chaperons make soluble 50% of your protein, sometime it is enough.

2) “we introduced 5 mutations in order to increase catalytic performance” Which activity are you studyng? If your protein disappeared you have also to consider that your mutations could generate a protein toxic for E.coli

ciao!

Paola

Dear Dr. Mopidevi, sorry for late reply. I don't pay attention on how much protein bind to the column before/after. My attention is only that my target protein is somehow dissapeared after passing the column. Thank's a lot for nice discussion and valuable suggestion.

Dear Paola, sorry for late reply.

about your question:

1. yes, I use Amp (for my gene) and Cm (for chaperone)

2. Thank you very much for your suggestion.

3. we works on haloalkande dehalogenase. About protein toxic, so far I don't have any idea.

ciao

Khomaini

according to my exp protein that get solubilised with help of chaperons degrade very easily. try to do a 1 day purification protocol & mix the sample with sds dye and heat & store . this will help you to trace after which step you are losing your protein