At 02:45 PM 9/25/97 -0400, you wrote:
>Has anyone out there had any success using anti-His tag antibodies for
>western blots? We have tried the anti-tetra and penta His MAbs from
>Qiagen for western blots of two different proteins which have C-terminal
>6xHis tags, and had no success. We are pretty sure the His-tag is
>intact on the proteins, given that the proteins were purified with
>Ni-NTA resin immediately prior to the western blot analysis. The Qiagen
>tech person suggested trying different blocking agents (BSA, powdered
>milk, and casein). We tried these, with no luck. Has anyone (not
>associated with Qiagen) compared Qiagen's anti-His Abs to those from
>other companies?
We have used both tetra and penta his MAbs for Western blot analysis. In
our hands,
pentahis is a better Ab and is able to detect at a lower level. We use ECL
for the detection system and incubate the mAb overnight at 4oC in casein or
dry milk. PentaHis has worked for a number of proteins that were N-tagged or
C-tagged.
With regard to other companies, we have tried the mAb from Dianova and I
cannot recommend that product.
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David Mueller
Department of Biological Chemistry
The Chicago Medical School
3333 Greenbay Rd.
North Chicago, IL
60064
Tel: 847-578-8606
FAX: 847-578-3240
