Which His-tag Ab for Western detection?

M. Johan Broekman, PhD mjbroek at mail.med.cornell.edu
Wed Dec 17 17:30:24 EST 1997


Hi All:

We have been unsuccessful at detecting my C-His-tagged protein
(glycosylated human recombinant) using ClonTech's or Qiagen's anti-His
MAb's, especially anywhere near the sensitivity levels the companies
claim.
We have been using a C-His HIVRT protein, purified via the Ni column
technique, as a positive control.  This was not detected by the His Abs
either, although an HIVRT-specific Ab readily detected this protein.
Any pointers for performing successful Westerns with His Abs would be
appreciated, especially successes with either ClonTech's or Qiagen's
Abs.

The Westerns were performed as follows:  Protein (lysates or culture
supernates, 0.5% serum) was SDS-PAGE separated, transferred onto
nitrocellulose, blocked with either 1% casein, or BSA, washed, probed
with primary Ab, washed, probed with biotin-labeled goat-anti-mouse Ab,
washed, and detected with HRP-streptavidin and ECL.

Thanks for any and all help,
M. Johan Broekman and Joan Drosopoulos





More information about the Methods mailing list