I am trying to detect a ~80kDa protein in HEK and Cos cell lysates. I use the purified GST-tagged protein as control and standards on the western blot. Last week, when I used 5 - 50ng of the purified protein for the controls, I got very clear bands. But this week when I ran the western blot again using the same proteins, I got very low signal. I am also getting some weird stains on the blot.
For the primary Ab, I use 1:200 and incubate overnight at 4 degree. For the HRP-conjugated secondary Ab, I use 1:6000 and incubate for 1 hour at RT. I rinse 4x 5-7min after each incubation, and use Pierce ECL kit and the LICOR system to visualize the blot.
I have attached one western blot from last week when it was fine, and one from this week where I could barely get any signal.
What could be causing this issue?