Immunoprecipitation and protein concentration

[Genecks]

I've been reading about doing immunoprecipitaiton. My first experiment failed really bad, not sure why. Anyway, I've been told I should have been doing BCA method quantification in order to determine the protein concentration of how much protein I have in a volume of solution. Well, I've been told that after taking tissue and lysing, I should do BCA. Afterward, do IP, but then don't BCA quantify and just move onto a western blot. Uh, why would I not want to know the protein concentration of the protein that I've immunoprecipitated out? Wouldn't that be what is relevant for observing what amount I've used in the western blot analysis?

[Genecks]

I've been reading about doing immunoprecipitaiton. My first experiment failed really bad, not sure why. Anyway, I've been told I should have been doing BCA method quantification in order to determine the protein concentration of how much protein I have in a volume of solution. Well, I've been told that after taking tissue and lysing, I should do BCA. Afterward, do IP, but then don't BCA quantify and just move onto a western blot. Uh, why would I not want to know the protein concentration of the protein that I've immunoprecipitated out? Wouldn't that be what is relevant for observing what amount I've used in the western blot analysis?

 

Nvm. BCA is done initially when generating homogenate and after IP'ing the protein of interest. It's useful for differentiating efficiency of initial cell lysis solutions when looking at protein concentrated of IP'd protein.

 

- Genecks

Edited April 3, 2012 by Genecks