Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Western blotting quantification and normalisation


  • Please log in to reply
2 replies to this topic

#1 julliver

julliver

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 08 December 2003 - 02:18 AM

Hello!
I would like to quantify signals from western blotting.
1. Should I repeat a sample (positive control) between all my gels?
2. What's the right way to normalise my data? Should I normalise in relationship with beta-actin (house-keeping) and also with my positive control?
Thanks for all inputs

#2 ramakn

ramakn

    member

  • Active Members
  • Pip
  • 17 posts
0
Neutral

Posted 11 December 2003 - 08:53 AM

You can quantify your western bands by gel densitometry. The software provided by Kodak is very good in this respect as it will also allow you to subtract the backgroundfrom the mean intensity. Good luck

#3 ros

ros

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 38 posts
0
Neutral

Posted 18 December 2003 - 01:54 PM

after you've probed for your protein you need to strip the blot and then probe again with a housekeeping gene (we use alpha-tubulin, but b-actin should also be fine). once that's done you can scan the films and quantitate them with a densitometer. normally i divide the value the densitometer gives me for my protein band by the value for alpha-tubulin for that sample. so that you end up with a ratio of protein:tubulin for each sample and then graph that. it's an arbitraty value, but it allows you to compare between samples. i dont think you would want to normalilse your data to your positive control.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.