My drug is specific to a nuclear protein. Trying to measure the intra-cellular binding affinity of drug to the target nuclear protein. I have tried to do the lysis of the cells using liquid nitrogen. The nuclear protein was not abundant after such physical lysis. Need help whether I can use any suitable lysis buffer? Can lysis buffer (e.g. RIPA or other types) denature the drug bound protein? Concerned about degradation of the binding between drug and protein if we use any lysis buffer..
You can isolate nuclear proteins by first isolating the nuclei only (separating nuclei from cytosol) and then extract the nuclear protein with buffer containing high concentration of salt (NaCl or KCl up to 1 M). You need to know the chemical nature of your drug as well, to know if or how the binding of you drug can be/is affected by the component of buffer you are using.
Thanks, actually we are trying to discover the intracellular binding affinity of our newly synthesized drug to its target nuclear protein. We found the binding affinity of our drug to the extracted target nuclear protein which was not intracellular or not in live cell. Now we are trying to get the information about drug stabilized drug-protein complex (i.e. ligand-protein complex) in non-denatured form. Can CER or NER kit degrade the binding of drug and protein? Could you please tell me, whether RIPA buffer can unstabilize or denature the ligand-protein complex or drug-protein complex? I meant will it be same?
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