I am purifying a protein from bacteria and don't seem to get anything from the Western blots using anti-6xHis. Unfortunately, even the positive control doesn't give a signal and I have used this (positive control) before and had positive results. What is happening, because the antibodies I am using are supplied and I am using them fresh?
Also, I realised, we also have anti-5xHis antibodies, can I use this to detect my protein which has 6xHis residues on WBs?
Hi Robert, if your positive control stopped working, it usually means that your assay is not working.
The worst thing that can happen to a biochemist (and all scientists) is to rely on an assay that you don't trust.
Hi there,
Did you check actually the transfer efficiency by staining the membrane with Ponceau Red before probing?
If you use Chemiluminescent method, the two solutions are easily gone wrong if someone forgot to change tip to take them in the past.
Hi Robert. If you decide to try a different antibody a can recommend finding a suitable antibody at www.pAbmAbs.com. Here, several different antibodies against His tag have been recommended for western blot, including this one
http://pabmabs.com/wordpress/?p=313
I hope this helps :)
kind regards,
Simon
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