I am starting to work with H9c2 cells. I will be attempting to transfect them with a tagged protein I want to study. I tried a pilot transfection, but when I harvested the cells in about 100 uL of RIPA + protease inh + phosphatase inhibitors, I got a really low concentration (0.2 ug/uL). I tried a western, but when I did ponceau staining, almost nothing showed up, so I can't assess whether the transfection worked because there's almost no protein. I seeded H9c2's in a six well and let them grow to about 70% confluency before switching to starve medium to do the transfection. I would estimate about 10^6 cells per well by the time I did the transfection. For lysis, I scraped the cells and lysed them with 100uL RIPA with rotation at 4C for 30 minutes. I'm wondering whether I should also sonicate the lysates. Any insights would be greatly appreciated.
Hi Ryan.
Firstly, I would like to ask you that is your protein familiar to your cell line.I mean the translation machinery of your cells would be possible to make the exact protein that you want.Then, It may be helpful if you check your RIPA,It should not be old and all of the pH must be correct up to your protocol.
Best Regards.
Hi Ryan,
I am using RIPA buffer to extract my protein. I do same what you said in your post only one thing I used to do which is sonicate my lysate. so I would recommend to soncate your lysate at least for 5 min then centrifugation at 11000 for 15 minutes then take the lysate without the pellet.
I would agree with Shahrooz Ghaderi, moreover, I would keep RIPA buffer out of light since it would potentially damage by sunlight.
good luck
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