Just remember that you are still required to dialyse the imidazole out for ethics reasons for the animal and have the antigen preparation in a suitable solution i.e PBS. If the starting concentration of protein is sufficient to remain within the protein assay working range upon its dilution, then this method will often reduce the amount of interfering substance in the sample to the point where it no longer interferes. I was worried for a while that the HIS tag would cause the titre to contain anti-his antibodies.but it didnt (well not that much that it detects non-specific His signals) and I now have three different antibodies prepared this way with great titre & specificity. The simplest method is to assay the sample after diluting it several-fold in a compatible buffer. In addition, I found the adjuvant-antigen mix to be "thicker" and easier to form than when doing it with a soluble antigen. Apparently in some cases the precipitated protein can cause a larger immuno response due to the inability of the host to remove the antigen in the same timely fashion as a soluble antigen. I solved the issue by using the precipitate for antigen injection into the animals anyway. In the end I couldnt dialyse the imidazole out without the protein preciptating. I had the same problem that you are now experiencing. Other protein for protein-protein interaction. Enterokinase does not work in the presence of imidazole. In one of the proteins, I have to remove His tag using Enterokinase and use the protein for antibody production. You may require higher salt to maintain suspension. Why are you dialyzing? what is the next step? if it is for gel filtration then you need higher salt to prevent nonspecific binding to the matrix.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |