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
- - - - -

Oscillation on my Western Blot results


  • Please log in to reply
3 replies to this topic

#1 titah

titah

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 08 June 2011 - 02:23 AM

Hi,

I would like to get some advice about Western Blotting technique. My research is concerned about A1 adenosine receptor. The results of my westerns are never stable…I’ve been observing an oscillation in the signal obtained! I got these three situations: strong signal, weak signal and no signal.

My protocol is as follows:

-Homogeneization of samples in 50mM Tris-HCl pH 7.4 with protein inhibitors.
-Samples were diluted into two volumes of a urea lysis buffer and incubated for 2 hours at 37ºC.
-10% SDS-PAGE gel, 92V (stacking) – 135V (running)
-Wet transfer, 350mA (2h30) for two gels
-Check transfer with Ponceau S stain (result: ok!)
-Block in 5% non-fat dry milk 1x PBS-TW (2h)
-Primary antibody incubation overnight in 3% non-fat dry milk 1x PBS-TW followed by 3x15’ washes with 1x PBS-TW
-Secondary antibody incubation for 2 hours at room temperature with agitation followed by 3x15’ washes with 1x PBS-TW
-The membranes were developed with chemiluminescent reagent ECL Plus

Any help would be appreciated,
Thanks in advance!

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,831 posts
415
Excellent

Posted 09 June 2011 - 01:21 AM

Fresh dilutions of the antibody each time?

fresh protease inhibitors each time?

#3 titah

titah

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 16 June 2011 - 12:25 AM

Fresh dilutions of the antibody each time?

fresh protease inhibitors each time?


Yes, since I've noticed oscillations on results, I prepare everything fresh and I'm still observing these variations...

#4 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,831 posts
415
Excellent

Posted 16 June 2011 - 03:59 PM

Perhaps it is something to do with the sample prep rather than the western as your protocol looks fine. Perhaps you are keeping some samples on ice for longer than others, or doing more freeze/thaw cycles?

As a note: You will sometimes see a difference in signal strength between two identical gels when blotting both in the same tank. I think this is due to some interference from one of the blots, as it always seems to be the blot closest to the black terminal that has stronger signal...




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

©1999-2013 Protocol Online, All rights reserved.