Blocking buffer for Infrared Fluorescence Detection in Westerns?

Dear Experts,

I wonder if anyone has tried to use common blocking buffers / blocking
reagents (BSA, gelatin, synthetical stuff) together with LiCor's
Odyssey system, or other, comparable machines that make use of
infrared fluorescence in the range of 600 to 800nm?

Would I get problems (that means increased background) with standard
BSA (0.5%, together with TX100 as detergent) on PVDF?

Just curious,

Wo

Hi Wo,

I have used the Odyssey system and found that PDVF produced high background.
There are special types that are supposed to be "low fluorescence" but I
didn't get to try them out. I quickly switched to nitrocellulose and just
stuck with that (I'm in a different lab now, so don't the Odyssey anymore).
This was ~18 months ago now, so I'm sure there should be some other PVDF
membranes that are designed for fluorescent detection.

I also found that using bSA as a blocking buffer produced a lot of
background so I used 5% milk. Where that failed I used the blocking buffer
from LiCor - it wasn't the best for the antibody that I was using, but I got
some OK gels after fiddling around with the laser and sensitivity conditions
on the machine. For all my antibodies I did use bSA in the primary antibody
incubation. I didn't try any other blocking buffers.

I also found that if I accidentally used Tween in my blocking buffer there
was higher background. (The manual says not to use Tween in the blocking,
but it is fine to use at all other steps, except for a final rinse without
it.) Taking tween out completely was not a good idea- horrible background-
it was accidental, but a lesson learned!

It look a little while tweaking the system, but following this general
protocol I had great success with this machine.

Cheers,
Amanda

On 27 Apr 2007 04:09:13 -0700, WS <novalidaddress@nurfuerspam.de> wrote: