3 replies to this topic

[indyslim]

Hello,

A quick check to see if anyone has tried any kind ELISA on cells frozen in DMSO, and then washed/lyzed etc.
How about ELISA or western blots on formalin fixed samples. I am interested in looking at phospho proteins in clinical samples.

Many thanks
Indy

[Ben Lomond]

It is difficult to know what effect the freezing process and DMSO will do to the protein that you are trying to measure without doing an experiment on unfrozen cells without DMSO in comparison with your DMSO treated and frozen cells.

As for the fixed samples...I don't think westerns will work, because formaldehyde is a cross-linker, so when you try to run your sample through acrylamide, I suspect it will just stay stuck in the wells because your protein of interest is stuck to everything else.   In terms of the ELISA, I think you will run into similar problems, and antibodies recognizing native phosphoproteins may simply not work on the fixed proteins which again will be cross linked. I am not sure that you will be able to specifically solubilize your fixed phosphoproteins in a reproducible fashion to get a robust ligand binding assay. An in-situ approach on sections might be preferable.
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[indyslim]

Thanks for your input, Ben. I agree that the results might be target specific and we might actully have to do a controlled experiment to get an answer.

Regarding fixation and ELISA/Western. I have heard multiple folks opine the same thing - crosslinking is going to make a western/ELISA impossible. However, I know of no one who has actually performed an experiement. Here is a reference that shows that you can still run westerns on fixed samples. (Journal of Proteome Research 2010, 9, 5188–5196). Again, like the previous case - this could be target dependent.

[AnetteM]

Hi
Interesting question. I have also read several articles were they have run ELISA on fixed cells so that should not be a major problem.
The western seems more porblematic however.
Bu I have not done any of the experiments my self.

It would be interesting to know how it works out if you try to do any of it.