Disappearing retard band

[Graham Atherton]

I have just run a mobility shift assay in which I compared adding 1.25ul
crude nuclear protein with 2.5ul (ul=ug). At 2.5ug I get a nice retarded
band which recognises the probe specifically (as judged by competition
studies). At 1.25ug input there is a similar band but it is maybe 1/10
to 1/20 as intense. I am clearly encountering some sort of cutoff
in binding kinetics - but what is it? I do not add carrier protein to
equilibrate total protein content between samples - maybe I should?
Does anyone have any experiance of this phenomenum?
Thanks
Graham Atherton
grggta at picr.cr.man.ac.uk