I'm going to extract total protein from 15 ml of a cancer cell line 24-hour conditioned medium. I need to know how much protein I can extract from this amount of CM, and is this enough for mass spectrometry analysis? And also which method do you suggest?
Roughly speaking protein content goes linearly with the cell mass and volume. Yield varies according to the cell line, hands on, extraction conditions, and how you optimize your expression. In general - Total protein that you can get from cell lines is like HEK-293 (200ug/1million cells), CHO (100ug/1 million cells), Hela cells (250ug/1 million cells), jurkat 50 ug/1million cells. They are approximate fig, such amount is good enough for mass spec. ( Note it is total protein, yield would be less for membrane prep )
Spectrophotometric analysis can be used to determine the precise amount of protein in cancer cells.
I think you want to look at the proteins secreted in the media, not the cell lysate, correct? If you have added FCS to the media, it will be difficult to analyze the secretome of the cells by MS because of the large amount of bovine proteins that will be present in the conditioned media, often much more than teh amount of protein secreted by the cells.
I would concentrate the 15 ml of conditioned media and control media that has not been incubated with cells down at least 10-fold and do a protein assay. Then run 20 ug protein on a gel. If there is no difference in banding pattern between the control media and conditioned media, then you will have to see if you can culture the cells in media without added FCS. Otherwise MS will identify bovine proteins and very few cellular proteins.
Thank you all for your answers,
Mary Cristine Charlesworth the conditioned medium I would use is not containing FSC or FBS. Do you have an idea of how much (ug) total protein you will extract from your 15 ml serum-free CM? Is it enough for MS?
Usually 1-2 ml is enough after 24 h culture. We load 20 ug protein on a gel lane, electrophorese, fix and stain, then cut out 3-6 sections from each lane (horizontally the same across all the sample lanes), depending on the band complexity. Each band is digested and the peptides analyzed by MS. We can identify many more proteins by fractionating this way.
I can’t say exactly how much protein your cells will secrete, but 15 ml should yield at least a few hundred ug, i think. Plenty for MS at any rate.
Thank you so much, your comments helped me a lot. Just one more question, from how many cells did you get 15 ml CM?
i don‘t know that answer because as a Core lab, we get the conditioned media from many investigators using different cell lines, and we don’t grow the cells ourselves. Sorry I can’t help with that one.
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