Here is some guidance. It is about drugs, but the same principles should apply to protein antigens. The recommended pH range for buffered vehicles is 4 to 8.

https://www.cambridgemedchemconsulting.com/resources/formulation.html

You also have to consider the adjuvant if you are trying to raise antibodies.

The isoelectric point may not be the reason the proteins are insoluble. They may not be refolded properly. You can also try increasing (or decreasing) the salt concentration to affect solubility, and lowering the protein concentration.

Here is some guidance. It is about drugs, but the same principles should apply to protein antigens. The recommended pH range for buffered vehicles is 4 to 8.

https://www.cambridgemedchemconsulting.com/resources/formulation.html

You also have to consider the adjuvant if you are trying to raise antibodies.

The isoelectric point may not be the reason the proteins are insoluble. They may not be refolded properly. You can also try increasing (or decreasing) the salt concentration to affect solubility, and lowering the protein concentration.

Dear Justin

the question is:

you would like to immunize mice with folded protein to maximize the probability to obtain specific antibodies also directed against conformational epitope or you think you are bit able to refold it or produce it as soluble changing domain, expression condictions or host and therefore you accept the risk to immunize with unfolded proteins and obtain a antibody response directed against linear epitopes that some times that reduce the probability to produce functional antibodies but sometimes work?

in the second case you have to discuss with the animal facility to undertand which is the maximun denaturing agent concentration that can be tollerated by mice (i think tgat each conutry has it guidelines- in italy for example in the past we were permitted to use freund formulation or urea but now it not permitted) in the past we performed immun nization with unfolded proteins replacing urea with arginine (1M) that guarantee the antigen solubility and its worked.

good luck

Manuele

Manuele Martinelli Dr. Martinelli, my dilemma is exactly as you stated "trying to maximize the probability of obtaining specific antibodies directed against the properly folded proteins" I am trying different refolding protocols, but the final problem will be finding an appropriate vehicle for mouse vaccinations

Dear Justin

to reply to your question:

if it is really the ph of the PBS the factor that induce protein precipitation during the dialysis. you can certanilly change the ph and try other buffers as acetate ph5, mes ph=6 or tris ph=8 that are different to pbs but not so extreme.

however i'm afraid that the precipitation that you observe is due to the fact that protein is not correctly refolded when you remove urea and in this case is possible that the change of ph will not produce better results and you have to optimize the protein sequence or the expression condiction to try to produce it directly in tte soluble fraction and not in tte inclusion bodies.

good luck

Manuele