I'm running EMSA and add the FAM Fluorescent Groups to 5' ends of my probe. However, the cis-element of the protein in the probe is close to 5' ends(only 7bp).Will the protein block the Fluorescence signal of the probe? I used Amersham Typhoon to camera the gel. I have confirmed the interaction between the protein and probe through yeast one hybrid experiment. And signal of free probe decrease when the amount of protein increases.
The fluorescence quenching by the protein is worth worrying about because it suggests that the probe is involved in the interaction between the nucleotide and the protein, which can result in an erroneous value of the affinity. It would be a good idea to change the probe oligo so that the protein binding site and fluorescent label are farther apart.
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