Protein analysis of conditioned media - How to prepare media before SDS-PAGE? (Oct/12/2005 )
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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.
Melissa
respond here or to rocknislets@yahoo.com
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