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Western Blot with fixed cells - (May/28/2013 )

Hi people,

is it possible to do a WB from cells that were fixed before (Cytoperm/Cytofix, contains PFA)? I am currently looking at literature and searching the internet on this topic, but I still wanted to ask you bioforumers for your opinion/advice/experience !

Thanks !

EDIT: Would an ELISA be a better alternative in this case ?


I don't think it would be possible after fixation ... instead they will be fine for FACS analysis eventually


Subsequent FACS analysis is not possible in this case, which is why we wanted to do a Western...


anyone else an opinion ?


One paper that might be helpful:

In it they fixed with 3.7% HCHO, 30 minutes and permeabilized with
0.1% Triton X-100 before sorting the cells, and then just made their
western lysates directly in SDS Sample buffer.

I've personally tried various fixation techniques prior to western blot, and most results are pretty horrible but depended on the protein.

Why do you need to do westerns? Can you do immunostaining? What about RNAscope technology?


Thanks for the reply, doxorubicin !
Hm, with immunostaining it's not really quantitative, I suppose... and RNAScope is rather for tissue sections I think ? Had been considering ELISA as an alternative though.