HELP! Western Blot: Anti His detection problem - Why cant my His tag be detected? (May/20/2010 )

Hi everybody!

To detect a His tagged protein on a WB I'm using a His-Tag monoclonal antibody from Novagen which is supposed to recognize 5 consecutives histidine residues regardless of the surrounding amino acid context. My protein has a 6His tag on the N-term position but it is not detected and I dont understand why. I used several positive controls with a Nterm His tag and again, no detection. However, a protein with a Cterm His tag was detected in my WB. But my antibody is supposed to have no Hi tag position specificity.....Do you have any possible explanations for that? (Im working on denaturing conditions so I dont think my His tag is stucked in the protein...).
Thanks in advance for your help.
Leen

We had problems with detection of his-tagged proteins on western blot using his-tag specific antibodies from several different vendors. I wrote to GenScript and asked them if they would send me a sample of their his-tag-specific monoclonal antibody, and it worked wonderfully, even against proteins expressed from pET16b, which attaches a 10x his-tag (GenScript's site only mentions their antibody's use with 6x, 5x, and 4x his-tags, so I asked them to let me try it out on a 10x-his-tagged protein before buying it, and they agreed).