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 and immunofluorescence results don't correspond to each oth


  • Please log in to reply
2 replies to this topic

#1 mik-i

mik-i

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 27 October 2009 - 06:34 AM

Ciao!

I am currently performing Western blotting on muscle samples. However, my results don't match for what I found earlier with immunofluorescence. For my protein of interest, in IF, I saw that it was upregulated in diseased tissue but that it was almost absent in normal control muscle tissue. However, in Western blotting on total extracts, both normal controls and diseased samples show the same amount of protein??? How can that be? The antibody is suitable for both IF and WB. I never had it before that something so different in IF (normal vs disease) is totally unchanged in WB?
Please could anyone help? I'm desperate :(

Tnx!
:)

#2 miBunny

miBunny

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 130 posts
1
Neutral

Posted 27 October 2009 - 05:54 PM

Do you know if you are working within the linear range on your western blots? Have you tried diluting your input lysate by 3 or 4-fold dilutions to see if the signal fades similarly?

Western blots have a very narrow linear range and it is very easy to saturate the signal. You need to be sure that you are using an excess of your detection reagent and it helps if you have purifed protein to run a standard curve with.

#3 mik-i

mik-i

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 28 October 2009 - 04:03 AM

Do you know if you are working within the linear range on your western blots? Have you tried diluting your input lysate by 3 or 4-fold dilutions to see if the signal fades similarly?

Western blots have a very narrow linear range and it is very easy to saturate the signal. You need to be sure that you are using an excess of your detection reagent and it helps if you have purifed protein to run a standard curve with.




Hi!

I'm afraid I didn't check that! I can't use the sample several times again and again as I don't have as much material (it's always a leftover of a patient's biopsy). So, I'm now doing a Western blot in which I'm at one hand using less sample and on the other hand diluting my antibody. So let's wait and see! Thanks already for the advice :rolleyes: !!! I also heared today that I could better do chemiluminescence, because then you can check the saturationgrade with certain software programs




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

©1999-2013 Protocol Online, All rights reserved.