Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protein Science > Western Blot Forum
Register Search Today's Posts Mark Forums Read

Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.

Mysteriously disappeared bands

Mysteriously disappeared bands - Western Blot Forum

Mysteriously disappeared bands - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.

LinkBack Thread Tools Display Modes
Old 02-02-2013, 12:03 PM
Pipette Filler
Points: 370, Level: 7 Points: 370, Level: 7 Points: 370, Level: 7
Activity: 0% Activity: 0% Activity: 0%
Join Date: Feb 2013
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Exclamation Mysteriously disappeared bands

Hi all! i am new here

i have read about bands that gone missing, so here is my very own mystery:

Repeating a WB experiment using the same recipes,tested antibodies etc as before, i reached the final step of developing the image out of my membrane. Using ECL for 5 min and then incubating another 5 min the membrane with the film into the cassette,I developed the image and it was ok; i saw my bands but there was background so i put my membrane into PBS-Tween (the recipe I always use) to wash it for about 30 min (5 min-washes in a total time of 30 min) in order to get a clearer image. The thing is, when I tried to develop the image again (ECL: 5 min, cassete: 5 min), all bands were lost, not a single sign. I changed ECL (truth is mine was old), but still not a sign of my bands, the film was clear. I changed films, fresh NEW (tested) ECL, I varied the time period of incubating the membrane with the film in the cassette (giving it more time), still not a thing (I gave it more than 10 shots). Finally, I incubate my membrane with aTubulin (tested antobodies first and secondary) and I tried to develop it (using my old ECL for 5 min and then incubate the membrane with the film for 2 min. Normally, atubulin bands appear HUGE after this period). I saw 3 very weak bands (though my samples were 6) so i incubated again the membrane with the film for 10 min expecting HUGE signal this time. When i developed it,... NOTHING, not a sign, not even the 3 weak bands.

So, is it possible that my membrane somehow "died out" during the PBS-T wash because after the washes I never saw my bands again ? I suspected the old ECL,too, that's why I used a fresh new one, but still there was no sign of my protein bands. And which is, after all, the best PBS:Tween? (I usually use e.g. 200ul of Tween in 200ml PBS but i have been told a great variety)

any ideas/have u experienced the same mystery?? sure thing, I am going to repeat the WB...
Reply With Quote

bands , disappeared , mysteriously

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
negative bands problem SallyG SDS-PAGE Gel Electrophoresis Forum 4 07-06-2010 08:03 PM
Mysteriously light S6 bands ajw7 Western Blot Forum 0 01-21-2009 04:08 PM
missing bands and ghost bands Manuel Gadin Protocols and Methods Forum 2 09-30-2008 02:12 PM
Increase Exposure, Bands Disappear?? Mediatorx Western Blot Forum 3 07-01-2008 09:18 PM
Help Migrating bands camherst Western Blot Forum 1 01-26-2008 02:48 PM

All times are GMT. The time now is 05:03 PM.

Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2015, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.11461 seconds with 16 queries