Protocol Online logo
Top : New Forum Archives (2009-): : Electrophoresis

HELP!- strange band appearing on immunoblot - strange band detected during blotting with anti-CTB antibodies (Jul/12/2011 )


I have been running SDS - PAGE gels on CTB containing samples and I have began seeing an issue that many lab mates are also having. It only occurs under denaturing conditions, ie. not during native electrophoresis, and it seems to be an issue related to the actual electrophoresis system rather than the samples, or stock solutions.
The issue is a heavy band in the 15-20 kDa range, we observe this when doing immunoblots on nitrocellulose membranes using Thermo Science chemiluminescent substrates, and cannot detect the same issue during staining with CoomassieBB. The band is not perfectly straight, and seems to bleed between the wells indiscriminately. The net effect is a heavy band from one side of the membrane to the other. This issue has not been seen during western blotting against other proteins, so it seems to be an issue specific to our anti-CTB antibody, yet it is not consistent in its appearence
-while the phenomenon may be seen with a certain set of samples, re-running the proteins has provided no such odd occurrence, suggesting the gel may be to blame.
-stock solutions (acrylamide, TEMED) have all been changed to new bottles, yet the issue persists.. All other components are prepared individually by lab members yet as many as 5 of us have had this problem
-We believed it could be an issue with our BioRad SDS-PAGE systems, yet upon identifying an electrophoresis apparatus that provided a clean, regular blot, re-using the same system the next day produced the issue.

Has anyone run into this type of occurrence? Our next plan of action is to borrow an apparatus from another lab, and to see if the same issue is observed. Also, we will transfer a blank gel to a membrane and blot in order to see if we can detect this inexplicable band.. Any other suggesstions on how to get to the bottom of this situation?

Any help is appreciated, thanks!
Attached Image
Attached Image

-Stew New-

so it is not the running front? nevertheless, it might be proteolytic fragments. Is it stainable with silver or India ink?

-Inmost sun-

thanks so much for the reply, but dont you find it strange the way it bled between the lanes and is even included in the lane containing the marker? furthermore, if it was a proteolytic fragment that was >15kDa i think CBB would stain in w/o issue..

-Stew New-

more so, some samples which had given the band before were run again and the band did not appear, so it cant be an issue related to sample preparation

-Stew New-