Hello, in your experience what is the best kit available in the market for whole cell protein extraction from human cells and gives a high yield. We are looking for a kit that will yield at least 4-5 mgs of total protein from 400 million cells.
The cells likely to be used are HeLa but suggestions for other cell lines that can provide such a high yield of protein extract will be helpful too.
Speedy responses greatly appreciated!
Thanks.
Dear Aditi,
I use many kits for nucleic acids... but never for protein! Is there a specific reason you want to use a kit?
Using standard RIPA (denaturing!) buffer to make whole cell lysates from mouse cells, I obtain a very good yield. In fact, I extrapolated my average yield to predict how many mg I would get from 400 million mouse B-cells. The result? 25mg. As HeLa cells are much bigger than my cells, I think you could expect to get greater than 25mg / 400million cells using RIPA buffer.
My protocol is as follows in case you are interested:
1. Wash cells twice with cold PBS and spin down gently (200g x 5min)
2. Resuspend in 300ul of RIPA (+ protease inhibitors) per 10 million cells and rotate at 4C for 30 minutes
>Observation, the DNA will be released from the cells and will form a white precipitate. This precipitate will be removed with a spin in the next step.
3. Spin 10 minutes at max (16,400g) at 4C for 10 minutes
4. Remove supernatant (whole cell lysate proteins) to new tube and Bradford
I'm attaching a standard industry (Thermo) RIPA buffer instructions as well.
Best of luck.
Thanks Nathaniel! Do you have a paper where this protocol/ method has been cited? That would be helpful.
Sure thing, Aditi!
I googled "RIPA buffer whole cell lysate HeLa cells" and found this decent impact paper (see link below) where they use this protocol to obtain protein from HeLa cells. Find their "protein harvesting and western blotting" section in their supplemental methods.
You can find lots of variants of this protocol. In some, you apply RIPA buffer straight onto plates of PBS washed cells (such as in the paper I linked down below, and in the manual I attached on my first post). In some, you first trypsinize cells, wash with 10% FBS (to inactivate trypsin with serum anti-trypsin factors), wash with PBS, then lyse in RIPA. I've read before that trypsinization chews up ~1/2 of cellular proteins, so it may be best to apply straight to the plates after PBS washing.
Good luck!
http://atvb.ahajournals.org/content/25/3/e10.full
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