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[VReinhart]

Hi all,

I've been running Western Blots for the past year using LiCor Odyssey Software and mostly using upright Applied Biosystems gel boxes. For most of the past year, I've been running 10-15 lane 8-12% Bis Tris gels without any real problem. For a new project, I've been running 20-lane 8-12% Bis Tris gels with 10 ug protein, 1:1000 primary antibody, 1:10000 secondary antibody and PBS 0.1% Tween for washes, as per prior optimization. With these larger gels, I've been getting patches of red cloudiness (for 680 secondary antibody detection) throughout the majority if my gels. I've noticed, however, that it doesn't appear for my positive control proteins - run on the same gel at 800 secondary antibody. Does anyone know how to get rid of this problem? Because it seems to be restricted to 680, I'm wondering if my secondary antibody is no longer working properly, but I'm fairly new to Western Blotting and am wondering if there is anything else I'm missing.

Thanks in advance for any help!

~Veronica