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!
- - - - -

Ghost and inverse bands and high background. Help!

ghost band high background beta actin galectin-3 c-jun

  • Please log in to reply
3 replies to this topic

#1 Elioelio



  • Active Members
  • Pip
  • 15 posts

Posted 11 October 2012 - 05:50 AM

I'm almost at my wits end with trying to get my western blots to work...

I keep getting ghost bands (inverse/white bands) where my protein should be. Also a very high background. 11.10.12 Gal3 30s.jpg 01010.12 c-jun 40s 2nd.jpg
  • My proteins are at 25ng
  • After running the gel I use Thermo scientific Superblock (T20 TBS) blocking buffer for 1- 1.5 hours, 12mls
  • Add 1:10,000 primary antibody. (I've used c-Jun, Galectin-3, Akt, JNK all from abcam)
  • 4degC on a rocker overnight
  • Wash x2 in TBST, then rinse in TBST 3x 5mins (sometimes maybe longer i.e total 30mins)
  • Add secondary antibody 1:10000 to 12mls TBST, 1 hour
  • Rinse and wash blot 3x 5mins
  • I use Supersignal west pico ECL. I blot the blot, then add 3mls on blot for 5 mins
  • Blot off the ECL, and image 1-40secs
I even stripped one blot yesterday that had ghost bands (cJun), and then added B actin antibody. Nothing came up on the image today 11.10.12 Bactin 5s.jpg

Any ideas at all I'd be grateful as we're a bit stumped! Possibly the antibodies are out of date? (2008). Although the B actin antibody should work fine.
Am I washing too much? Too much protein? Too much antibody?

#2 PhDinAcronyms



  • Active Members
  • PipPipPipPipPip
  • 37 posts

Posted 11 October 2012 - 03:34 PM

Ghost bands often appear when you have way too much antibody, as the HRP rapidly depletes the ECL reagent in the region of your bands. Also adds to the high background. I would try even less antibody and you could also try using a less sensitive ECL reagent.

And you're definitely not washing the blots too much. 3x washes is the absolute minimum number of washes I would do to get a nice blot with low background, so I would add a few more washes as well.

#3 cbf88



  • Active Members
  • Pip
  • 14 posts

Posted 12 October 2012 - 11:38 PM

load more and get fresh antibody unless those were aliquoted and frozen

#4 Elioelio



  • Active Members
  • Pip
  • 15 posts

Posted 15 October 2012 - 04:04 AM

Hi thanks for the replies. I actually found that my TBST stock was at the wrong pH - was at pH4! So I'm made it fresh. I repeated the B actin and it worked perfectly, but I also repeated Galectin-3 and it didn't - still got ghost bands. I will try much less antibody today - maybe 0.8ul in 12mls.

We were worried that maybe the antibodies just don't work anymore. I aliquoted and froze the Galectin-3 antibody, but it was aliquoted at 4ul - so each one has been taken out 3-4 times. All the other antibodies I've used are kept in the fridge and not aliquoted...

Also tagged with one or more of these keywords: ghost band, high background, beta actin, galectin-3, c-jun

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

©1999-2013 Protocol Online, All rights reserved.