Hello, has anyone had issues with EMSA data making little sense and rando behavior, I have recently been trying to run EMSAS of a 62Kda protein, with a High PI of 9.09 and I am experiencing very random behaviour as in some times the free DNA in the no-protein control sample has very little signal compared to when I add my protein, and also the free DNA completely disappearing say when I add 40 molar excess of protein and then the free DNA re-appears at 50x of protein, does anyone know what could be going on? i simply need to quantify the free DNA for Kd measurements since I do not expect the complex to enter the gel. With this behavior of the assay however it is impossible to get a Kd value. I have tried higher pH of 10.2 in CAPS page buffering system and agarose gels, but the issue remains, I also added BSA to the reaction so the total protein concentration is 0.1mg/mL and that did not eliminate the random behavior. I have changed the reaction volume from 6.25ul, 10ul, and 16uL but the issue remains, I also lowered and raised the DNA amount from .01 to 0.3pmol in my reaction. I have used low protein binding tubes as well was sigma Cote on the tubes but the issue persists? Any advice for this issue? I am using a FAM labeled DNA substrate and detecting the signal via a typhoon imager. I attached some gel images to show what I am dealing with. Thank you