Currently doing some Western blots. One of my blots looks completely fine when I probe it for human SNX27 with clear bands, but when I reprove it for GFP it's impossible to quantify any bands. It think the problem is a lot of background non-specific binding, but I don't know how best to fix it or why it only seems to be a problem when I probe it for GFP. For context, the samples are just HEK293T cell lysate. I've used this GFP primary antibody for a while and never had any issues, and when I do a similar experiment the blot looks completely fine when I probe for GFP. Membrane was blocked for 1 hr in 6% milk PBST.