I can infer difference in proliferation and migration of my cells when subjected to condition media. but when I isolate proteins and run on PAGE gel am not seeing any difference in protein bands. I have even tried concentrating my media with speed vac.Still I see no difference. One set of my samples have serum and rest are serum free. Has anyone worked on conditioned media with serum? I feel proteins are not getting separated because of the albumin.
My suggestion is instead of checking overall protein pattern, check specific proteins in presence and absence of serum by Western blotting technique. For example it is well established (we have also checked) that serum starvation stopped the cell cycle at the G1 phase of the cell cycle. One of the reasons is synthesis of CDKs and some other proteins involved in the cell cycle will be completely going down after serum starvation. So, check these proteins by Western blot after serum starvation
The particular growth factors and chemokines that you may be interested in are probably such minor components of the medium protein that you can't see them just by SDS-PAGE, especially in medium with serum, which is mostly albumin. I agree with Tapan Mukherjee that you should, if possible, focus on specific proteins of interest and use Western blot or ELISA to measure them. For medium with serum, remove the albumin first with a column designed for that purpose. These are commercially available.
If there is an unknown but very minor protein that you wish to identify in the medium, that is a more complex problem.
Why don´t you do a 2D gel electrophoresis??? Yo can obtain a global vision o protein patern.
I did try 2D. But apparently something was wrong with the strips, so I was wondering what other techniques can I possibly do until new strips arrive.
You can use LC/MS for checking the protein expression in two different condition.
OMG! Yes, something was wrong. Did you clean samples??? DO you want to do the analysis of secreted proteins?
Thanks Mr. Kannan and Tomas. Ya am interested in secretome analysis. So am isolating proteins from media. Some media samples I work on has serum. Doing LC-MS might give me huge number of peaks. I might not be able to differentiate the peak and noise as you know serum's composition of proteins is undefined. I was told that you shouldn't subject a mixture of proteins to MS. Pls correct me if am wrong.
Hi,
I have attached the paper where u can get the protocol for the secreted protein analysis.
I hope it will help you.
Serum is a problem, why don´t you deplete it? You can eliminate the most abundant proteins from serum samples.
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