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Protein quantification from paraformaldehyde-fixed cells


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#1 victorius

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Posted 13 November 2009 - 12:47 PM

Hello,

Does anyone know of a protocol to extract and quantify proteins (e.g. by BCA or Bradford assays) from attached, PFA-fixed cells?

Thanks

vic

#2 amelia417

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Posted 15 November 2009 - 02:47 PM

Hello,

Does anyone know of a protocol to extract and quantify proteins (e.g. by BCA or Bradford assays) from attached, PFA-fixed cells?

Thanks

vic


You should still be able to lyse the cells with a lysis buffer containing protease inhibitors without the PFA disrupting the lysis process. I would do this if you're dealing with small wells. Add lysis buffer with the plate on ice and put the cells on ice on a rocker for about 30 min. Check to verify lysis under a microscope and then transfer the cells out of the wells and measure protein according to the usual protocol. Remember to use a very small amount of lysis buffer that is proportional to the number of cells, or you will be diluting the lysed cells too much and will be outside of your standard curve for measuring protein. Alternatively, if you are using larger plates, it may be easier to just scrape the cells in lysis buffer using a rubber policeman. Do this on ice.

#3 victorius

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Posted 16 November 2009 - 08:22 AM

Thanks so much Amelia,

I tried that - it's basically what I do for non-fixed cells - but the protein content (BCA assay) in PFA-treated wells was about 1/4 of that measured in similar, unfixed wells. I used RIPA buffer + inhibitors in both cases (BTW, do I need protease inhibitors after PFA fixation...?). I think the lower protein content is due to remaining cross linkage in pFA-treated cells... I'm trying now adding 2%SDS to RIPA and boiling the lysate, I read this worked for paraffin-embedded tissue.

v/




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