I have a doubt and difficulty interpreting my tryptophan fluorescence graph since the concentrations do not seem to be creating a pattern in the curves.
It would be expected that in larger concentrations, such as 1000nM, would have a higher quenching when compared to 800nm and 300nm for example (Figure B). But by looking at the graph we see that the opposite occurs (Figure A). The fluorescence was done in triplicate and similar results were observed.
Anyone have any idea what might be generating these curves without patterns?
Protein: DHPS
Ligand: Pteroic Acid
The data was collected as follow:
Signature=ISS_Experiment_Ver_ 1_0
Product=Vinci2
SoftwareVersion=BETA.2.1
BuildNumber=322
AcquisitionType=Photon Counting
AcquisitionFormat=L
AcquisitionSide=Right
Measurable=Intensity
MaxIterations=10
RightEmissionWavelengthBandwid th=8
ExcitationWavelengthBandwidth= 8
Visualization=Y:Intensity,X: EmissionWavelength,PlotType:2D
EmissionWavelength=type: numeric,unit:nm,from:300.0,to: 420.0,step:1.0
ExcitationWavelength=type: numeric,unit:nm,fixed:295.0
Space=EmissionWavelength
Columns=EmissionWavelength, Intensity,IntensityStdError
There is no trend in your data between the concentration of quencher and the amount of quenching. You need to reevaluate your experimental technique to find out why there is so much variation. Once you get that sorted out, make sure you account for inner filter effect by pteroic acid and/or intrinsic fluorescence of pteroic acid, if any.
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