Protein Isolation of very low cell number... - (Jul/03/2008 )

Hi guys,

i want to isolate total protein after a few days of culture. Per dish are around 100k cells and i am not able to get enough protein with RIPA. Is there any other buffer (or a kit) around that could help me with this issue. I can not get more cells because it´s a primary culture of neurons.

Any suggestions...thanks a lot
Tobi

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we use in such a case a 3x conc lysis buffer using only 20-30 µl;

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RIPA is quite strong buffer. I don't think your problem is related to getting all protein out, it is that you can not have more starting material. No buffer can help there.

1. As B. said, try to minimize ripa/well, and run everything. However, keep in mind that at lower buffer volume, you need to make sure that you are covering and lysing all cells.

2. If it does not harm your particular protein, you should do freeze/thaw cycles (2 or 3), after adding a little RIPA. That would bring out all protein.

3. Alternatively/additionally you can also sonicate your little volume in order to increase the released protein amount.

4. Use very low detection range kit for western (Pierce supersignal dura, ECL pico etc.)

..

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Have you considered using a flow cytometry analysis?

100,000 cells per well should be enough...

And if the protein concentration is low you can amplify the fluorecent signal by double labeling...

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i also had the same problem while handling primary CLL cells. best way is to start with a high cell number, and later sonicate the cells in the least volume possible(this makes sure that u get both nuclear and cytoplasmic extract) and of course increased protein content.

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