6 replies to this topic

[lalalalove3]

Hello everyone!

So after transferring, I cut the PVDF membrane so that it would fit into a case in which I could block with 5% milk. However, I guess I was too slow (it was my first time), so the membrane dried out and I was able to see the lanes. I just quickly put my membrane into the case and it is now blocking overnight at 4 degrees in 5% milk.

Do you think that everything is still going to be okay? How might this affect my results? Please help me! Thank you for your time.

[rustyshackleford]

Hi lalalalove3,

Don't worry, I think it will be fine. Actually, I once went to a western blot seminar where a speaker recommended allowing the membrane to dry after transfer and before blocking because it allows for better protein detection. I don't know if this is true (I've never tried it myself), though I don't see why it would cause harm.

Best of luck with your first western!

[HomeBrew]

rustyshackleford, on 02 September 2010 - 06:30 PM, said:

Actually, I once went to a western blot seminar where a speaker recommended allowing the membrane to dry after transfer and before blocking because it allows for better protein detection.

I was told this at a seminar, too -- from a Millipore technical rep, I believe.  I haven't tried it either...

[laurequillo]

Yeah sometimes after transfer you can see the bands, but dont worry, it should be ok

[lalalalove3]

Awww, thanks everyone. I'm really grateful for all of you who answered. I am not as worried anymore.

[mdfenko]

in theory (i've never tried it), if you let pvdf dry after transfer and don't reactivate with methanol then you don't need to block. no more protein will bind to the membrane. antibodies will only be able to contact the protein bands that are already on the membrane.

[Acehunter]

In my labgroup, we let the PVDF-membrane dry after transfer before we stain the membrane with the primary antibody diluted in milk. IT works like a charm..

Ace