Developing polyclonal antibodies

bsnmin at yahoo.com bsnmin at yahoo.com
Thu May 6 23:49:37 EST 2004


Hi,

I have a problem. I have a mouse protein cloned in pET 28a vector and over expressed in E.Coli BL21DE3 and protein purified using NiNTA-agarose column. But I have serious problem in folding this protein as it autolyses into several fragments (we know that it is not a protease nor we acquired no protease contamination during its purification). Further, the protein is highly non-immunogenic and we encountered serious problems in injecting crushed acrylamide gel bands, for raising the antibodies. Can any of you suggest if there are any methods for dissolving the protein precipitate in non-ionic (or denaturing) solvents and use it directly for raising polyclonal antibodies. What about use of solvents like DMSO, Glycerol, PEG, CHAPS, NP40, Tween20, glyxol and what is affect of these solvents on the antibody production? I do not have much idea about the protein solubilizing ability of these solvents also. Perhaps some of them may dissolve hydrophobic proteins.

Thanks.

Dr. B.S.N.Murthy
Scientist; CCMB; Hyderabad; India



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