Diffusion of Antibodies in PVDF and NZ membranes

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Tue Mar 20 18:10:49 EST 2007

Stop fighting! It's just about westerns. Not religion, abortion,
soccer and other things people people usually are smashing each
other's heads and become terrorists.

In most cases, at least in my experience, westerns are quite resistant
against anything and the standard procedures as you may find them in
any tech manual or datasheet will do. Of course, they're optimized not
only for good results but rather for good sales, especially when they
suggest you to use a new aliquot of antibody every time.
Naturally, there are special proteins that won't stick well to either
NZ or PVDF or both, so you'll have to do it faster or change the
conditions. I'd recommend changing (i.e. normally increasing) the salt
concentration in the buffers. Adding ethanol or methanol to the buffer
(as you probably do it during the electroblot) also might help, but it
could denature your antibodies and / or kill your detection system. It
must be a pain to find out (how does one do that???) that your
favorite protein does not like the standard conditions. Probably most
people conclude first their prep was bad or that the ab would not

Returning to my initial question, I want to give you some more details
an tell you my experience from the past few days: I'm using westerns
to test for protein phosphorylations in 60 human biopies which are
very very limited samples. I must get most out of it. Two gels were
run and blotted in parallel in order to treat all the samples in the
same way to make the results comparable. O course, they also are
probed with the same ab solution and the ECL is done in parallel. I
now have lots of small stripes (one gel makes six membrane slices
corresponding to different molecular weights) of a PVDF membrane where
always 2 strips go into the same 50 ml tube which contains a primary
AB dilution. By some sort of minimizing of surface principle, these
membrane strips (of course 2 always have the same size) have a
tendency to stick together after some time. One can't control the way
how they do it (face to face, back to back, face to back and vice
versa). Surprisingly (the incubation takes place on a rocker in the
cold room over night), it does not seem to matter much, as I have to
say after several days of analyzing these westerns. Either the
incubation time is long enough to allow for the diffusion of the
antibodies (the package of the membrane says 45um pore size, but I
believe that's a typo, it rather should read 0.45um, but these holes
still should be big enough to let proteins diffuse through) or there
is enough time for the membrane to reach the equilibrium before the
cohesion happens (thus I was lucky) or the cohesion is not strong
enough and loosens from time to time or there is simply enough liquid
and antibody between the sheets.

Best regards,


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