Protein analysis of conditioned media - How to prepare media before SDS-PAGE? (Oct/12/2005 )
I would like to investigate the proteins beings secreted from encapsulated cells. For this, I am going to prepare conditioned media at various time points for SDS-PAGE. Initially I plan to just use 1D gel electrophoresis & mass spec. As the project fine tunes itself, I plan to continue on to 2D-gel electrophoresis.
Can you suggest the best way to prepare a total protein sample from conditioned media? Another group in our institute is using TCA/acetone extraction for the same type of result.
We are not looking for a specific protein at this point in time. We are just wondering what the cells are secreting during culture.
Thank you kindly. Anything you can offer would be helpful.
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I do not have exhaustive experience in this, but I was going to suggest TCA/acetone precipitation as a starting point. Be aware that the media will also contain some protein, so perhaps precipitating some unused media would be a worthy control...
Thank you kindly for your prompt response. I was planning to have a "control media" sample as well - so your suggestion reinforced my original thinking. Thank you. I guess I will go ahead and try this TCA/acetone precipitation. Thank you once again. Melissa
Good luck! Let us know how it goes!
Maybe its too late to reply to this topic but now I am thinking of analyzing conditioned media as I believe that the protein that I am trying to find in whole cell extracts is acively secreted. I jus found some protocols about precipitating my hormone with antibody and then with protein A agarose
Could some one tell me if I am in the right track? and if so, do you think I can store the media until the analyiss at -80'?
thanks in advance