I'm running an EMSA and I'm finding that adding additional protein causes the free probe band to start to disappear instead of shift up into a new band. It seemed to work before so I don't really understand what the problem might be. I've attached a picture of three gels. Each gel is basically probe 1 being exposed to increasing concentration for 7 lanes and probe 2 being exposed to increasing concentration of protein for 7 lanes. Probe 1 shifts into a faint blurry band but probe 2 just seems to shift slightly and mostly just disappear. What could be the problem?

For the mix I'm loading with the values I know so far.

6.25uL 4x buffer (80 mM Hepes-KOH ph 7.8, 400mM KCL, 4mM EDTA, 40% glycerol) reading off these concentrations so not sure

.0125uL or 0.05 dna ss 309ng/uL

1ul BSA

variable H20

variable protein

2uL labeled probe

The gel itself is

20mL 0.5x tbe

11.5mL H20

2mL 50% glycerol

6mL acryl/bis 40% 37.5: 1

probes are about 30bp

It takes me about 15ish to 20 minutes to add the protein. I add the probe and let it mix with the protein for about half an hour. I tap vortex for about a second. Loading the gel takes about 20ish to 30 minutes or so. Gel is prerun for 30min. The gel is run after loading at 110V for 2hr.