I have a protein, I am expressing in CHO cells ans it has the 6 His tag. I did the western blots and there is so much unspecific binding. The primary antibodies are raised in mouse. Could this be the reason for the unspecificity. I used dilutions of 1:10000, 1:25000 and 1:50000 (L-R) for the anti His Ab and 1:2000 for secondary Ab.
Additionally, anti-peptide Abs against a portion of the N-terminal of my protein seem to give two band of lower molecular weight.
What could be the reason?
NB: Lysates were done in duplicates using RIPA and 2x Laemli and the molecular weight is about 60 kDa.
The 2 bands seen in the second blot may be proteolytic products of your protein.
For the top blot, did you run a secondary only control? It's worth doing because that would help narrow down where your problem is coming from.
The bottom blot may be degradation products of your protein, as Adam says. I assume you would have used protease inhibitors in your lysis buffer but if this was not a fresh prep, they may have lost their activity. If you want a clear answer and have the facilities in your department, you could excise the bands and use mass spec to see if they are indeed fragments of your target sequence.
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