I tried to detect the endogenous expression of my protein of interest, but I can't. However, at the same time, I can verify the overexpression of the protein with different antibodies.
I tried to change my cell lines and tested four different antibodies, but it didn't work.
Could anyone give me some advice?
You also try to (IP) your protein. IP enables you to isolate your protein of interest from as much lysate as you want and second thing I don't know which kind of antibody you use if you use polyclonal antibody ?? try to use monoclonal antibody
Hi,
Possibility is that your endogenous protein expression is low enough for antibody to detect or its not present at all or its under control which is not allowing it be expressed in the cells.
1) Run a gel with different amount of protein lysate and be sure to remove DNA by sonication.
2) If at any point protein is detected by Ab then you can keep it as standard amount to detect that particular protein that particular cells.
3) If that wont work then same thing need to be done as Ab titration.
Hope it will work for you.
Thank you
Hi Mahsa,
Try to increase the amount of total protein up to 40ug (micrograms) per well. And see if that helps your antibody to reveal band(s). Generally, monoclonal antibodies are good to use. However, if you found one that works best to detect the overexpressed protein of interest, I would focus on that one and try to use other methods of Ab validation like IP pull down, IHC, ELISA.
How about mRNA expression of your target gene, how much fold is it different from the overexpressed proteins?
Also, if its cell membrane protein or nuclear, I would try to use a protocol of cell membrane or nuclear proteins extraction.
Good luck with your experiments.
Selim
It sounds like the protein is being expressed, but it is not being expressed in the correct place or at the correct time. This could be due to a number of factors, such as a mutation in the gene that codes for the protein, or a problem with the promoter region of the gene.
Protein overexpression causing mutations in genes that code for proteins, or the presence of oncogenes. Protein overexpression can lead to a number of different diseases, including cancer.
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