how to detect antigen of low concentration? - (Mar/30/2007 )
I get big problem now, the concentration of the protein i want to detect is very low. I can hardly find its band by western blot.
what should i do?
I would perform an immunoprecipitation
I am having the same problem now. Can anyone suggest a method to precipitate small amount of proteins?
My proteins can be dissociated from cell membrane in a buffer, but the dissociated proteins are so less that I don't see it well after 40min exposure (in WB). If going longer than that, background starts to show in the blot. Another thing is that I cannot decrease the amount of treatment buffer.
I don't know if there is anyway except western blot I can compare the basic quatity of my protein with that under different circumstances?
You could also think about just precipitating the lot with ammonium sulphate If you want it native) or even acetone or TCA for denatured protein, then resuspending in a small volume and running that.
the solution of swanny sounds good.
It's easier to perform than an immunoprecipitation.
However, if your protein of interest is expressed at a very low yield, you might have to load too much protein on the gel, and then the migration is bad.
however I would try first the TCA precipitation before the immunoprecipitation.