I am doing EMSA using LightShift Chemiluminescent RNA EMSA Kit from Thermo Scientific. For the detection, I use stabilized Streptavidin-Horseradish Peroxidase Conjugate and Chemiluminescent Substrate (Luminol/Enhancer Sol and Stable Peroxide Sol).
I have been having this problem on and off, even though I perform the experiment with fixed protocol. I cann't figure out why sometimes I have black backgroud and white free-probe signal as shown in the picture.
By the way the bands shown in the picture is not the band we expected.