On 5/6/04 11:49 PM, in article 20040507044937.32699.qmail at ww02.hostica.com,
"bsnmin at yahoo.com" <bsnmin at yahoo.com> wrote:
>>> 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.
First, a few questions back:
What species are you raising the antibodies in?
How homologous is your protein to its homologue in the species you are
immunizing?
What were your "serious problems" when using the acrylamide gel bands?
"Serious problems" is not enough information for others to help with
troubleshooting.
Did you only immunize with crushed gel or did you emulsify it with another
adjuvant?
I am not clear on why you want to use alternative solvents to keep your
protein denatured for immunizations. You could also immunize with the
insoluble protein. You've already lost the protein's native conformation by
denaturing it so you might as well just try the insoluble protein in a
common adjuvant. With no data on the effects of other solvents in a vaccine,
you may just waste a lot of time, money and protein.
Stacy
