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

Molecular Biology Techniques Molecular Biology Forum. Includes forums for common molecular biology techniques.


Patchy Western Blots (particularly anti-phosphotyrosine blots)

Patchy Western Blots (particularly anti-phosphotyrosine blots) - Molecular Biology Techniques

Patchy Western Blots (particularly anti-phosphotyrosine blots) - Molecular Biology Forum. Includes forums for common molecular biology techniques.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 02-27-2013, 07:30 PM
Pipette Filler
Points: 23, Level: 1 Points: 23, Level: 1 Points: 23, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2013
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Patchy Western Blots (particularly anti-phosphotyrosine blots)



Hi all,

I have been having occasional problems with my western blotting where I get large patches with no signal at all and then lots of background everywhere else (attached is an example of a anti-phosphotyrosine blot).

I don't believe it's a transfer problem as I have done a fast green stain and I can see bands of protein on the entire membrane.

I don't think it is an ECL problem as I have reapplied ECL and the same patchy patterns appear.

I don't think it is a problem of the distribution of secondary antibody because if I rinse the membrane with TBST and then incubate again with secondary, followed by ECL the same patterns appear.

I feel it could be a primary antibody problem (maybe the primary antibody is just not getting to those patchy parts of the membrane?) but if I rinse off the ECL, reapply primary, secondary, and ECL, I still see the same patterns.


As this doesn't happen to me all the time, only sometimes, I feel I am doing something technically wrong. No one in my lab has seen this problem, and someone has watched me perform the western blot and can't seem to figure out what my problem is.

I'm hoping someone has seen something like this before and can give suggestions.

Thanks!
Attached Images
File Type: jpg ptyr fail.jpg (8.6 KB, 0 views)
Reply With Quote
Reply

Tags
inconsistent , patches , western blot


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
Normalization protein for Western blots? Peter Frank Protocols and Methods Forum 8 01-04-2012 12:52 PM
"Center effect" in Western Blots Nattylad Western Blot Forum 1 11-01-2010 07:54 PM
Antibody Reuse for Western Blots? IoDiNE Western Blot Forum 1 08-26-2008 10:24 PM
Stripping western blots Ann Sutherland Protocols and Methods Forum 2 10-26-2007 12:36 PM
Special treatment required for dried Western blots? paul_wary@yahoo.com Protocols and Methods Forum 4 05-18-2006 10:06 AM


All times are GMT. The time now is 02:36 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.12842 seconds with 17 queries