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-   -   Loading control for secretory proteins in culture medium (http://www.molecularstation.com/forum/immunoprecipitation-forum/73083-loading-control-secretory-proteins-culture-medium.html)

deeps 06-02-2010 05:34 PM

Loading control for secretory proteins in culture medium
 
Hi,

I am doing the immunoprecipitation of secretory proteins from culture medium and looking for a loading control for these secretory ptns.My P.I was telling that erk is a membrane ptn and I cann't use it for my purpose.So does anybody have any idea about it?

Thank You

MJD 09-06-2010 04:38 AM

Re: Loading control for secretory proteins in culture medium
 
Positive control cell lysate (the cell from which the proteins are being secreted)
-note there should be some of the protein of interest still inside the inside the cells

Negative control either media or cell lysate from cells without the protein of interest

MJD 09-06-2010 12:30 PM

Re: Loading control for secretory proteins in culture medium
 
Sorry. I was confused on what you were looking for, but it seems that you need a protein to serve as the loading control. This will vary depending on the cell line, since you were not more specific. You should use Google Scholar or PubMed to find a secretory protein that will be secreted from your particular cell line and that will not vary based on different treatments or conditions. Good Luck!

alien!!! 01-27-2011 04:01 PM

Re: Loading control for secretory proteins in culture medium
 
Quote:

Originally Posted by deeps (Post 420758)
Hi,

I am doing the immunoprecipitation of secretory proteins from culture medium and looking for a loading control for these secretory ptns.My P.I was telling that erk is a membrane ptn and I cann't use it for my purpose.So does anybody have any idea about it?

Thank You

Hi there,

me too, I would like to do an IP on conditioned medium. I'm planning to incubate the medium with my cells for a couple of days, concentrate the medium with Amicons and then do IP...

My Question is... Do you add FCS to the medium in which you collect your secreted protein??? Does it not interfere with IP?

I would be grateful about an answer, thank you!

John


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