As per my research, drives me to inhibit a particular gene which codes for a protein involves in multiple functions.
After treating the cells with my designed drug against that protein, i have found the expression of that particular mRNA got reduced by Quantitative PCR.
But i want to confirm it at the protein level.
Since the target gene is novel, there is no commercial antibody available to it right now?
Is it possible to find the protein level by any other staining or non-antibody mediated method like fusion of GFP to the target gene etc.,?
since raising an antibody against that protein cost (in lakhs) which i couldnt afford it right now.
kindly help me in this regard!!!!
If you know the size of the protein then yes you can check for its presence or absence using western blot. just run the blot with known protein weight standards.
Hi there,
if you don't have any specific antibody available then epitope tagging is a solution using anti tag antibody for probing WB membrane.
I am not sure whether tagging will solve your problem because your drug is obviously working on the expression of the protein (reduction of mRNA). In this case you would need a construct with the original promoter and the tagged cDNA. No possibility of a functional assay?
If the expertise and equipment are available for it, the iTRAQ method of LC-MS-based proteomics can be used to differentiate the levels of proteins present in 2 samples treated in different ways.
If your protein has a specific activity, then try to measure its lowest effect using the specific substrate, this might help you to prove the low number of protein molecules in your treated cells. Good luck.
How presence of a protein can be measured has been outlined by the collegues nicely above, but absence is difficult: "The absence of evidence is no evidence for absence"!
Following Adam's and Tichaona's advice. I think you can do both. If you know the size of the protein you can identified it through western blot and actually take the band for analysis at LC\MS\MS method. You can quantify it's amount and to be sure that this is your target protein you can sequence it (through LC\MS-MS- just your band).
Regarding Erik's note... You have to do the experiment in different concentrations of the drug. In this way you will have a decreased amount of the protein relative to your control.
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