Wet transfer: Western Blot

[tech123]

Hi..This is my first time doing wet protein transfer. I use PVDF membrane. Run at 70 V for ~ 3 hrs. After transfer my membrane becomes little dry and the outer coat of the membrane starts to come out leaving dark spots on the membrane. Also do protein ladder go bad? They have started to appear very dim in gel. Any suggestions!!
Thank you

[mdfenko]

we need more information.

3 hours is a long time to transfer unless the protein is very large

what is the pore size of the membrane (since it's pvdf i assume that you activated it prior to transfer)?

transfer buffer?

what do you mean by "outer coat" of the membrane? the membrane should not be layered (unless you are using supported nitrocellulose).

yes, protein ladders can go bad, if they are stored improperly. are the bands faint before transfer?

a picture would be useful

[tech123]

Thank you...the problem got solved after charging my membrane with MeOH.
This is kind of silly but I am trying to detect three proteins A, B and C. Unlike Immunohistochemistry, Why i cannot detect all the proteins in a single membrane when the antibodies against my protein are raised in different animals? I know the size of my proteins.

[mdfenko]

Why i cannot detect all the proteins in a single membrane when the antibodies against my protein are raised in different animals? I know the size of my proteins.

because you will require three different secondary antibodies.
you can do it all together on one membrane since you know the sizes of the proteins probed for are separated sufficiently to distinguish one from the other.

[relaxin]

Yes, you can use a mixture of antibodies on one blot, just use a mixture of second antibodies or Protein A+G-conjugated HRP. But if you have not done Western blot with these antibodies before, you may want to play it safe by incubating with one antibody at a time. Some antibodies may give you multiple bands.

If the proteins of interest are quite different in size, stripping is not necessary. Just re-block and probe with another antibody.

[eafaidley]

Depending on the MW of your different proteins, A, B and C, you could also (to keep it clean) bisect the membrane horizontally using a razor, to then probe each respective piece with the appropriate antibody depending on MW range of each piece.
Make sense?
Good luck!