Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protocols and Methods Forum
Register Search Today's Posts Mark Forums Read

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Blocking buffer for Infrared Fluorescence Detection in Westerns?

Blocking buffer for Infrared Fluorescence Detection in Westerns? - Protocols and Methods Forum

Blocking buffer for Infrared Fluorescence Detection in Westerns? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 04-27-2007, 11:09 AM
WS
Guest
 
Posts: n/a
Default 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

Reply With Quote
  #2  
Old 04-29-2007, 11:00 PM
Amanda Beardsley
Guest
 
Posts: n/a
Default Blocking buffer for Infrared Fluorescence Detection in Westerns?

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:
Reply With Quote
Reply

Tags
blocking , buffer , detection , fluorescence , infrared , westerns


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
protein purification magnificientleo Proteomics Forum 11 12-29-2010 09:01 AM
How to make blocking buffer from Whatman Kit LauraG Antibody Forum 1 05-14-2010 03:57 PM
SDS-PAGE Protocol moleculardude Protein Science 14 02-12-2010 11:27 AM
Production of Monoclonal Antibodies - Technique in Mouse Cellbiogal Antibody Forum 2 01-08-2009 10:52 PM
detection of beta-nitroaniline via fluorescence? John Hines Protein Forum 0 10-22-2003 07:05 PM


All times are GMT. The time now is 09:45 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.12154 seconds with 16 queries