Western blot detects exogenous protein but not endogenous - (Apr/12/2015 )
I am trying to detect endogenous protein for my gene of interest. There is only one commercial antibody available. Also, there is limited literature on this gene and two papers state they were not able to detect endogenous protein with their in-house antibodies targeting the same region of the protein as the commercial antibody. I have overexpressed this ORF in a primary cell line with low mRNA expression using a Dox inducible system and can detect exogenous protein on my Western blot even with low Dox induction.
In regards to Western blot detection of endogenous protein:
1. I have loaded 75ug protein from total cell lysates harvested from two human cancer cell lines with the highest mRNA expression
2. Primary antibody 1:200 and secondary 1:2000
Under these conditions I should be able to detect the endogenous protein if present. Is there anyway to increase my sensitivity or enrich for my protein? Or could it be a protein stability issue? The protein acts in a repair complex for DNA damage response. Maybe I need to stabilize the protein by UV or etoposide treatment? Or use a nuclear extract? I just think it is strange that I can detect low exogenous levels (induction with 50ng/ml dox) but I cannot detect endogenous. Why would there be differential stability of exogenous vs endogenous levels?
I would appreciate experiment suggestions for detecting endogenous levels by Western blot.
How do you know that the Dox induced is low expression? Most Dox inducible systems are under the control of a viral promoter, which produces quite strong expression even with low levels of Dox. These systems are usually on or off, there isn't much in between.
1. I previously used this system for different gene and titered the Dox concentration in a primary human cell line comparing it against primary cells, immortalized and cancer cell lines with a wide range of mRNA and protein expression. The Dox inducible expression can be fine tuned to be within biological range. Also, I can see a step wise increase in expression with increase in Dox concentration. Additionally, I compared the Dox inducible expression against a vector that uses a constitutive CMV promoter to drive expression. The Dox expression is many magnitudes lower at the highest concentration as compared to the CMV driven expression. This can also be shown quantitatively with the Dox inducible system driving firefly luciferase.
2. In regards to this gene, I also compared my Dox inducible system against vector that uses a CMV promoter. I saw the same thing. I still need to compare the endogenous mRNA expression in the cancer cell line vs the Dox inducible mRNA expression.