I am trying to detect endogenous protein for my gene of interest. There is no commercial antibody available for mice. I have tried to detect the protein of my interest with the home made antibody generated by my lab however this antibody can only detect exogenous one and not working in endogenous one. Is this the problem with the protein stability? Why would there be differential stability of exogenous vs endogenous levels?
I would appreciate experiment suggestions for detecting endogenous levels by Western blot.
There are several reasons why your antibody (monoclonal or polyclonal) couldn't detect your protein of interest:
- It's not there or not produced in high enough quantity in your cells or tissue samples (degradation could be one reason for this, but not likely, if you denature the sample fast enough there should be something detectable)
- It is modified (glycosylated, phosphorylated, etc) in the eptiope that is recognized in the exogenous protein (what exactly is that exogenous protein? recombinantly produced or from a different source/organism?)
- It could be present as an isoform where the epitope(s) is(are) missing or incomplete
Check for mRNA first to make sure it is expressed in the sample. If you have access to Mass spec try to find the protein because this is usually more sensitive than Western Blotting
Also, it may not recognize the denatured structures. Test the affinity for native antigen in elisa by plate binding it.
Dear Sya Retno
You did not mention in which host you expressed the protein. You should also explain more about your work method. Explain a little about the nature of your target protein so that I can guide you better.
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