Transfer problem - Loss of MW markers after transfer! (Jun/29/2007 )

Hello everyone!

I used to tranfer to a semi dry apparatus with 1x tris-caps transfer buffer at 10V for 30min and got a good transfer (checked by coomasie and ponceau)
But recently we decided to change the transfer buffer to increase the efficasy of our transfer (because we thought that our protein of interest might not be efficiently transfered) so we used:
Cathode buffer: tris caps, 0,01% SDS (60mM Tris, 40mM CAPS, 0,1%SDS)
Anode buffer: Tris caps, methanol 15% (60mM Tris, 40mM CAPS, 15% methanol)
And...after trasferring for 30min at 10V the MW markers (and I suppose some of the proteins) were blown through!
I tried again for 15min at 10V and again the MW went through again (I could see them on the back of the bottom whatman paper)
So I'm afraid that since I'm loosing the markers I'm loosing some of my protein as well (the ponceau staining is "lighter" comparing to my first tranfers with only Tris CAPS buffer)
Any ideas?
Is it OK if I use again 1x Tris-caps again without SDS and Methanol?

Thank you in advance!

-charis-

hi,
if you are using PVDF membrane, then you need to activate it by dipping in methanol for 1 min.

Good luck

QUOTE (charis @ Jun 29 2007, 06:01 AM)

-veerubiotech-

Thanks,
but I neglected to mention that I'm using nitrocellulose membrane...

-charis-

QUOTE (charis @ Jun 30 2007, 01:23 PM)

Thanks,
but I neglected to mention that I'm using nitrocellulose membrane...

To check if your transfer worked, you could stain your membrane with amidoblack or ponceau s. This was also wery helpful for me. Staining is routinely performed when unstained markers are used.

-moljul-

OK guys ...
I figured it out...
First of all I didn't soak the nitrocellulose membrane in water before soaking it in transfer buffer (as of course it was mentioned in the membrane brochure!). That way I did not allow the membrane to "form its hydrophilic layer" and that way making it less efficiant in binding hydrophobic molecules...etc, etc
Secondly I found out that adding SDS in the cathode transfer buffer was too much making the proteins run a lot more efficiently. The SDS concetration in gel, lysis buffer and sample buffer is enough.
Combining those two together..and...voila!...I got again a decent transfer.

Sorry for my little panic attack and thanks for your attention

-charis-