Our lab usually uses RIPA buffer, but mostly on total cell lysate and mouse tissues. I want to isolate proteins in cell culture media, but I am not sure if using RIPA buffer is ok?
I also saw some references for using TCA to isolate proteins, but I cannot find a good protocol for cell culture media.
Hello Kristen Navarro
Using TCA to isolate proteins would be an appropriate method. You may use the protocol provided in the link below for cell culture media.
https://www.its.caltech.edu/~bjorker/Protocols/TCA_ppt_protocol.pdf
But you need to separate out the floating cells, dead cells and cellular debris by centrifuging the culture media at 900× g for 10 min at 4 °C so that the soluble proteins remain in the supernatant and the other unwanted components (cells) are pelleted down. Then proceed with the protocol given in the link above.
Best.
Malcolm Nobre - Thank you for this protocol! Can the final product be stored? If so, what should the pellet be resuspended in, and at what temperature should the sample be stored?
Hello Kristen Navarro , yes, the final product may be stored until further analysis.
You may do so by resuspending the pellet in 1-2ml of PBS and 50µl of 8% phenylmethylsulfonyl fluoride (PMSF) (protease inhibitor), and store it at -20°C.
Best.
Mohamed Khashan - I am asking about isolating proteins from cell culture media, not the cells
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