I have been running native page for FAM DNA substrate ( fluorescence samples) for protein DNA binding reaction. Binding is there but towards the end of the lane , I am loosing signals drastically.
I have checked my protein for nuclease contamination and it is nuclease free protein.
I am unable to figure it out , at times the signals are lost in the middle lanes also.
I am giving extra attention to pipetting, so I know that is not the problem.
I have attached the image.
If the dark band in the photo represents the fluorescent DNA, then the loss of the band represents binding to the protein, which results in a complex too large to enter the gel. It remains in the well and gets lost when the gel is removed from the electrophoresis unit.
A lower percentage gel may allow you to see resolve the DNA-protein complex, at least at the lower protein concentrations.
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