ELISA with cell lysates?

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Sat Apr 3 03:14:52 EST 2004


Peter Frank wrote:

> I have done ordinary quantitative Western blots in replicates but I
> assumed ELISAs to provide me with more accurate and precise results.
> 
> Would dot blots give even better results than ordinary quantitative
> Western blots in replicates? I have never done protein dot blots
> before, only performed cDNA dot blots so far.

Dot blots can be used to quantify proteins with very good
reproducibility and a linear relationship of signal vs protein amount
over 5-6 orders of magnitude (ELISA only one order of magnitude).

Use a 96-well vacuum manifold (reusable ones are available from BioRad,
Millipore sells the disposable stuff), in either case high-protein
binding PVDF membranes should be used. These trap > 95% of the proteins
in the sample (nitrocellulose only 60%). If exact quantitation is not
required, spot blots can be made by spotting 2 ul of samples directly
onto a membrane with a pipette.

Pipett your samples (and standards) into the wells. I have made good
experience with using native proteins (diluted with PBS as appropriate),
but a recent pubication suggests that even higher yield may be obtained
by denaturation with 15 volumes of 40% methanol, 3% TCA and 2% SDS.

The resulting blots are developed like Westerns, using HRP-coupled
secondary antibodies. The chemoluminescence is counted in a 96-well
chemoluminescence counter (e.g. Berthold). 

Note that the high dynamic range is a consequence of the direct
counting, if you use photography instead, you will be limited to about 1
order of magnitude, just as in ELISA.

Compared to Western blotting you loose the info on molecular weight of
the protein which binds the antibody, hence you have to check with
Western that you get binding only by one protein of appropriate MW.



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