How to Immunize with Inclusion Bodies?

[uas2]

Hey i have a problem. Ive got my protein in inclusion bodies unfortunately , and I wonder if I could just purify and use them for subcutaneous immunization of rabbits?

Can one alternatively solubilise the includion bodies in 6 M urea and use that solution directly to immunize rabbits to make antibodies?

I also wonder if the same could be used for intraperitoneal injection in mice to get monoclonal antibodies.
cheers

[admin]

Hey there,
I don't think this is the best approach. I would try to clear your proteins from the inclusion bodies (ie purify more). You don't need much protein just fairly pure protein so you don't waste time in rabbit care, housing, and poor things getting unnecessary bleeds. I would look into getting your protein secreted (using a tag) in yeast and just purify litres of the stuff from media...

just my opinion maybe someone else can chime in here?

[swanny]

I'd agree with admin. Best thing is to change your expression to get soluble protein. Look at expression temp, [IPTG], tags. Only work with the IBs if these have all failed. IBs are useful because they are fairly pure to start, apart from proteins that hang on to the edges; you need to get rid of them first.

To start with, you want to clean up the IBs, but going straight to 6M urea will be too harsh. Wash them in 1M urea, then 2M urea (ie, wash and spin, then separate the pellet from the S/N). Run pellet and S/N on a gel, as well as the untreated IBs. You'll see many of the contaminants will wash off into the S/N.
If you have to go up to 3 or 4M urea, but the IB might start to fall apart.

Once the pellet is pretty clean, then you can hit it with 6M urea to dissociate the whole thing (unless it has already done so). Remove the urea by dialysis or dilution into a large volume of buffer and purify your protein.