Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > General Science Forums > Biochemistry Forum
Register Search Today's Posts Mark Forums Read

Biochemistry Forum Discuss and post questions regarding the study of Biochemistry. If you need homework help this is the place to ask!


western blot bands too strong

western blot bands too strong - Biochemistry Forum

western blot bands too strong - Discuss and post questions regarding the study of Biochemistry. If you need homework help this is the place to ask!


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 10-11-2012, 07:47 PM
Pipette Filler
Points: 423, Level: 8 Points: 423, Level: 8 Points: 423, Level: 8
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2012
Posts: 4
Thanks: 0
Thanked 0 Times in 0 Posts
Default western blot bands too strong



Hi guys,

I am attempting a western blot, and probing for beta-tubulin as a loading control. I loaded 40ug of total cell extract per lane, and the bands for my protein of interest look great. Unfortunately, when probing for beta-tubulin the bands come out too strong. I am exposing it to x-ray film and it is becoming saturated even for a 1 second exposure (I can see my bands glowing on the membrane in the dark room). I diluted my primary and secondary antibodies 1:2500, and I suppose I can go to 1:5000 if necessary. Any tips would be appreciated. Also, can I simply wait for the HRP to use up more of the substrate to get weaker bands? I was thinking that stronger bands might use it up faster and appear weaker than those that are not as strong to begin with (if that makes any sense), which would be a problem. And how long does it generally take to use all of the substrate (minutes? hours?). Thanks!

ICS
Reply With Quote
  #2  
Old 10-22-2012, 06:17 PM
Kneeanderthal's Avatar
Pipette Filler
Points: 68, Level: 1 Points: 68, Level: 1 Points: 68, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2012
Posts: 26
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: western blot bands too strong

Load less (maybe 20 ug) when probing for beta-tubulin and dilute HRP antibody 1:5000, even 1:10000 (had the same problem with an anti-strep HRP). You could wait but I've seen the HRP active for days even after removing the substrate.
Reply With Quote
  #3  
Old 11-20-2012, 09:57 AM
Pipette Filler
Points: 66, Level: 1 Points: 66, Level: 1 Points: 66, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2012
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: western blot bands too strong

We dilute up to 1:25000 secondary antibody when we do beta-actin. You could also try diluting your ECL detection reagents. We've found Milipore's to be much stronger than Amersham's and needs to be diluted more when detecting our loading control proteins.
Reply With Quote
Reply

Tags
bands , blot , strong , western


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
Western blot with more strong bands biotom Western Blot Forum 3 09-08-2011 01:49 PM
Western Blot Mystery - Developed bands don't adhere to membrane cyrus Western Blot Forum 4 01-20-2009 09:08 PM
Way too many bands on western blot es0teric Western Blot Forum 1 09-19-2008 11:54 AM
Western Blot - Extra Bands moleculardude Western Blot Forum 3 10-17-2006 03:24 PM
Nobel Prize for David Thomson?! caltechdude Physics Forum 428 01-21-2005 10:39 PM


All times are GMT. The time now is 10:16 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 0.12429 seconds with 16 queries