Protocol Online logo
Top : Forum Archives: : Protein and Proteomics

Random Background Noise for Western Blots - I'm getting noise only in some lanes (Jun/13/2005 )


I was wondering if anybody has run into this problem I just had today. I ran some blots for process waste streams and came noticed I had a whole lot of background show up for some lanes and not others. For example,

Gel 1:

Lanes 1-6, which had the in process samples only showed 2 bands representing my target protein.

Lane 7, my control standard was basically all noise (basically band after band showed up)

Gel 2:

Lanes 1-5, was basically all noise for the in process samples (although lane 6 didn't have much noise but that sample was at a much much lower concentration)

Lanes 7, showed the correct 2 bands (my control)

I prepared both blots at the same time and treated them the same way. The control was prepared in one batch as well and just split up for each gel. As far as I can tell, I did the same exact thing for both blots.

Does anybody know why my samples had noise and the control was fine for one blot while the complete opposite occured for the other blot? I've been looking around and I've seen overloading as the cause of background..which the low concentration lane being fine suports. But both my controls were the same concentration and total protein yet it had different i'm confused now.. Any help would be appreciated. Thanks



If this has just started, after previous successful runs, I would suspect contaminated glass plates or a bad batch of nitrocellulose or a bad batch of blocking solution.