I did a western blot analysis. There were two different proteins in the samples. One protein is His-tagged and the other does not. For the western blot analysis i used an anti his antibody as the primary antibody. On the Westerblot you can see the bands of both proteins, although one protein does not have a His- tag. How can that be?
It might well be your secondary antibody which directly binds to the other protein. You should try different dilutions of your secondary antibody
Jean-Pierre is right. But you should also be sure, that your anti-his is not to old or contaminated. Maybe there is a lab mate who could help you out with some µl of his/her anti-his. And for anti-his, you also should use a proper dilution. The dilution recommendation in the data sheet is not always useful, but may serve as starting dilution. For exempl, if you started with 1:1000, than also try 1:5000. And also your protein sample: try to dilute the sample before loading the gel. This also might help.
How specific is your anti-his? Is it to 4, 6, 8, 10 his sequence? Some proteins have natural runs of histidines that can be recognized by anti-4his antibodies
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