Hi,
Has anyone here has done condition medium from cancer cell line and run the samples for LCMS?
I have done it by culturing the cells in the medium and then starving(serum-free to avoid the abundant of albumin). My method is i collected the condition medium and freeze dry, then i proceed with TCA percipitation and finally quantite the protein using micro BCA.
when i run lcms, suprisingly i've got like 20 or less protein from that condition medium(secretome). And the secretome that i've got are not very significant to be related with cancer pathways or gene.
Could somebody help or give me an opinion regarding this?
Thank you and i really appreciate.
Hi Nazilah
1. You got to be careful using "serum free media" for any cell line as it may put cells into a stress and you will end up having secretome unrelated to particular cancer cells. I would suggest using media supplemented with transferrin, BSA ( 0.1 %), EGF, FGF, Insulin, hydrocortisone etc.
2. You need to decide on which time point would be good enough to give you a secretome, not contaminated with proteins leaked from the dead cells. Therefore before collecting the condition media, you may want to perform LDH assay to find find out the percentage of cell death at 24h, 48h and 72h.
3. In best of the cases, precipitation of complex mixtures (of proteins) may just yield 30-60% of total protein. Therefore, I would suggest to perform LCMS directly on condition media rather then precipitating it. You may also want to do a tryptic digestion before injecting a sample.
4. Alternatively, you could run a gel, loading a same sample in multiple wells, silver staining the gel and cutting the bands across the gel in a way that you would be able to collect two fractions based on the molecular size (Remember, lesser the complexity of protein mixture, i.e, lesser the number of proteins , better the resolution of LCMS data). In such cases, one need to do In-Gel tryptic digestion.
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