I am interested in proteomic analysis of glioblastoma spheroids (diameter 200-300uM).
How many spheroids should I grow to get atleast 3-5ug protein at the end of lysis?
I will use M-PER containing phosphatase and protease inhibitors lysis buffer (Thermo Fischer) for lysing the spheroids.
Hello Simar Pal Singh
This is a tough question to answer until you actually perform the lysis on a certain number of spheroids. Decide on growing a certain number (I don't know, perhaps 10,000?) then lyse and measure the protein concentration using an assay such as Bradford or BCA. Perform this lysis and measurement with multiple replicates then you will have the answer to how much protein a certain number of spheroids will provide.
I did a quick back-of-the-envelope calculation: if we assume one HeLa cell has a diameter of 30 µm and total protein content of 150 pg, then to get 10 µg of total protein we would need approx. 67,000 HeLa cells. If your spheroids have a diameter ten times larger than HeLa of 300 µm (and making the BIG assumption they therefore have 10 times more protein per cell), then you may need approx. 6,700 spheroids to get 10 µg of protein.
Good luck!
Best,
Peter
Hi Peter
Thanks for your response and thinking along.
Indeed, I will try it out as it can also differ per cell line.
Simar
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