I am working with 6XHis-tagged proteins for Ninta pulldown assays. I
subcloned a library of genes in this His vector that I generated by
oligocloning of Met-6Xhis encoding oligos. So, the His is at the
N-terminus and just 6XHis. Unfortunately, I have severe troubles to detect
my proteins with either a penta-his AB (qiagen) or a AB against 6XHis (Santa
Cruz, 16-002). I know that the cloning and expression is OK as the pulldown
works fine and I can detect the protein by the AB against the protein. but
for most proteins in my library i do not have a specific AB.
It seems that most people generated a larger tag that contains some
additional amino acids to generate a stronger epitope. We have a good his ab
that recognizes c-terminal his-fusions (it requires a his-cooh) but this is
not functional for this library of course.
Is there anyone out there that knows of a good working 6XHis AB which also
recognizes plain 6XHis fusions at the N-terminus of the protein of interest.