Completely agree, you need a higher DNA concentration. We have had luck with 10 nM DNA.
Also, bubbles have a HUGE impact on how the fluorescent signal is measured. Make sure you spin your plates down (assuming you are using them) to remove any bubbles. We just had an undergrad read his anisotropy assay and the data looked horrible. He then realized he had not spun his plate down, after doing so the data was much more consistent. Are you doing technical replicates? We do at least triplicates per plate. All the best, Morgan Morgan E. Milton, PhD On Fri, Jul 21, 2017 at 9:48 AM, Opher Gileadi <opher.gile...@sgc.ox.ac.uk> wrote: > FP is the ratio between two fluorescence measurements; if the fluorescence > signal is too low, you will still get a ratio but it will be essentially > noise. Try to perform the measurements at 10-50 nM DNA. If your binding > affinity is in the low nM range, you may have to use other methods to > measure KD. >
