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.
I don't see the free probe alone but I think that here you have 100% binding. High amounts of extract may degrade by contaminated nucleases
It used to happen when the protein has lower binding affinity with probe. This results into smearing kind of band shift.
The experiments cannot be interpreted as there is no free probe lane as pointed out earlier. In addition, at least one lane has to contain an assay of "cold" oligonucleotide competition; the best is to titrate the probe at molar ratios 1:5, 1:10 and 1:50. A positive control is highly recommended to start with to assure that everything works. Generally, EMSA patters differ if nuclear extracts or recombinant proteins are used. When using recombinant proteins expressed in E.coli, cold oligonucleotides needed to detect the competition are at ratios 1:50 or even more.
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