Hi all,
I have problem and i don't know where i am making mistake. I am working with cancer cell line MCF-7 stable transfected. So I am doing simple experiment just to check protein expression. One day I plate cells next day i collect proteins and i perform protein assay. But when i do protein assay results are not matching in sense that concentrations of proteins are different and it supposed to be same, because i plated same number and it can not be big difference in cell growth for 24h. Any suggestions what can be a problem?
Plating cells could be uneven, and despite all efforts their growth conditions will vary
a bit between the wells. When I had to do assays in 96 wells on A431 cells, it required 6 replicates just to obtain stdev of 10-20%.
Situation might be helped by normalising your activity of interest to something related to viability/amount of the cells ( WST assay?).
which buffer you used for protein extraction. Did you digest them well.
But is it possible that in 24h they have such a big difference in cell growth, its same cell line, just different clones? I am afraid that i am making some mistake in plating cells, or collecting proteins. But i am doing same thing for a long time and i never had this problem before.
Thank You very much Misha
We are using cold lysis buffer (50 mmol/L Hepes, 150 mmol/L NaCl, 1% Triton X-100, 5g/ml pepstatin A, 5 g/ml leupeptin, 2 g/ml aprotinin, 1mmol/L PMSF, 100 mmol/L sodium fluoride and 1 mmol/L sodium vanadate, pH 7.5).
After Collection I check all plates under microscope, and they are empty.
Thank You Irfan
They should not grow much, but they might have different metabolic activity.
Try not to stack the plates so every well would experience the same temperature regime.
I would check some control protein/enzyme with ELISA or enzymatic reaction.
- Could your cell line contain minor contamination? (this way you plate 1000 cells
that may or may not contain 1 contaminating cell)
You can try to purify protein from clones before cell plating. If you will still see the difference in the protein levels you will know that the protein amounts in both clones vary and this is the reason for you results. Actually, you can collect cells from both plates at different time points , when each plate reaches confluence. From full plate you should have ~ 400-500 ug of total protein
Thank you very much for answers, i will try to do what you suggested. Yes MCF-7 are clumping very much, but i think i am dispersing them well because when I count i can see if they are clumping or not.
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