Recently, I try to extract the native form of proteins (for immunoprecipitation) from insoluble fraction (cell lysis pellet).
How can I do?
What kinds of lysis buffer are better?
*Experimental steps
1) cell lysis using Triton X-100 lysis buffer
2) after centrifugation, collected soluble fraction and insoluble fraction (pellet)
3) Extract native proteins from pellet using XXX
Under regular cell growth and protein production you do not have native protein as as inclusion body but misfolded protein. If your protein of interest is part of (or the whole) inclusion body you need to dissolve it with urea (8M) and then run a folding procedure.
I quite agree with Omar about using 8M urea to solubilise your protein. You can also use 6M guanidine HCl to resuspend the pellet. This will perform similar function to 8M urea, however guanidine HCl doesn't have the downside of forming isocyanate which could react with some side chain residues of your protein. The denaturation can go on for 1-3hours with continuous stirring. After which you will have to dilute the 6M urea protein mixture. This can be done by either slow dilution or fast dilution method. The European Molecular Biology Laboratory offer some practical guide on how to extract misfolded protein from inclusion body.
https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/invitro_denaturation_refolding/index.html
https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/solubilisation_renaturation/
Hi
please check following protocol:
http://www.currentprotocols.com/WileyCDA/CPUnit/refId-ps0613.html
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