I have get insolube protein at 1mMIPTG after 4 hours induction time. How can I find solube protein for purification by his tag nikal colum.
Hi there,
Before trying optimization of culture condition, I would make sure the His tagged protein is actually expressed by WB using anti Histag antibody. If it is the case (ie. expression of insoluble protein), I would lower temperature, lower IPTG concentration in order to slow down the process and therefore favor the folding of the protein. Other aspects to explore: chaperonin coexpression, use of autoinducible medium.
Hi there,
Before trying optimization of culture condition, I would make sure the His tagged protein is actually expressed by WB using anti Histag antibody. If it is the case (ie. expression of insoluble protein), I would lower temperature, lower IPTG concentration in order to slow down the process and therefore favor the folding of the protein. Other aspects to explore: chaperonin coexpression, use of autoinducible medium.
Thank you very much to response, I already have anti his tag Ab for WB, but I do not use it until now..
Did you see the induction? Please load a gel with uninduce and induce sample. What vector you are using. Are you expressing protein of bacterial or eukaryotic origin?
First confirm the expression as mentioned by Dominique liger and Paul lesbats.. with anti HIS ab then go for the purification..
Hi Hassan,
You should first confirm your protein by Western blot; then, purify it. If your protein has large molecular weight, and you want it in a native status (non-denatured), you can try this solution "B-PER Complete Bacterial Protein Extraction Reagent"
https://www.thermofisher.com/order/catalog/product/89822?SID=srch-srp-89822
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