In article <48sqos$fno at fiona.umsmed.edu>, hutchins at fiona.umsmed.edu says...
>>M K Archer (fr80 at dial.pipex.com) wrote:
>: We are trying to develop a western blot protocol without using
>: SDS-PAGE and electroblotting.
Yes, protein dot blotting. Its been around for years.
The method involves sucking protein
>: through a nitrocellulose membrane in a 96-well dot-blot machine (usually
>: used for DNA/RNA dot-blots). The antibodies are also added into the
>: wells and sucked through the membrane.
>Whoops! I think the problem is sucking the primary and secondary antibodies
>through the apparatus. Antibodies are proteins, right? What's to
>prevent them from adhering to the membrane just like the antigen?
Careful! The "best" method is to allow the antibody solutions to percolate
through the membrane (no suction). The vacuum should really only be applied
when using the washing solutions. The antigen and antibody *do* require some
time to attach to the membrane (they don't have any of that nice SDS to help
them to stick there).
>: Our problem has been we are unable to block the membrane
>: sufficiently. The secondary antibody binds to the membrane even if the
>: primary antibody is not present. We have tried a variety of blocking
>: agents (BSA, Superblock-Pierce, etc.) but without success. Has anybody
>: tried this sort of experiment or does anybody have any ideas.
>After binding the antigen (sample) to the membrane, remove it from the
>dot blot apparatus, dry it if you wish, then treat it in a floating bath,
>roller bottle or Seal-A-Meal bag, just like a 'real' Western blot.
It depends on what you are trying to assay. If you are titreing your antibody
then I'd suggest sticking to ELISA. Dot blots are usually used for
quantification of antigen as this requires the higher binding capacity of
For quantification of antigen, apply the antigen in the apparatus and
wash. Then dis-assemble, block, chop up the membrane if necessary (if you
are using several antibodies/dilutions) and develop as per a normal protein
If you are trying to titre your antibody then coat the entire membrane
in antigen (single concentration) and block before insertion into the
apparatus. Trying to block in the apparatus can be very tricky so varying
concentrations of both antibody and antigen on the same blot is difficult.
I've never had much trouble after blocking with 3% BSA in PBS.
However, this is after optimisation of the concentrations of primary and
secondary antibody - too much antibody will make any blot look messy, no
matter how much you block it. What detection method are you using? - ECL
tends to increase the background. It may also help to try some other kinds of
membrane - what are you using at the moment?
Oh yeah, I'd also put in a suggestion for trying a slot blotter
instead of a dotter - the edge effects in a wide dot blot well make
quantification a little tricky.
...and of course, you *have* checked the specificity of your primary antibody
before using it in dot blotting... ;-)
Good luck with your dottin'
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)