Hello i am fairly new to the field of physical chemistry and only have a vague memory of it from undergrad classes.
my FRET assay is set up like a regular FRET assay, so the enzymetic activity of my protease gives off fluorescence signal that i detect using my 96 well plate fluorescence meter.
I fully understand the whole idea of Michelis Menten equation, however, i am having trouble converting the "raw" fluorescence signal to the reaction rate or velocity (product concentration/time). For example, if my reading is 5000 Fluorescence how would i convert that 5000 Fluorescence unit into velocity? would appreciate if someone can use imaginary numbers to guide me through the process.
Would appreciate any help thank you.
To get the fluorescence intensity of the substrate when it is fully converted to product, you can either have the 2 products synthesized (costly, but a really good idea), or you can use a high enough enzyme concentration to convert all of the substrate to product.
A complication arises if the substrate concentration is high enough that the inner filter effect becomes significant. The inner filter effect is a reduction in fluorescence intensity caused by absorption of light by the sample itself. This is generally insignificant at nanomolar concentrations, but can become significant at micromolar concentrations. It will be detected as a sub-linear increase in fluorescence intensity as a function of concentration.
Fluorescence intensity is proportional to the amount of product.
You need two things: fluorescence intensity of the unreacted substrate; and fluorescence intensity when fully converted to product.
Now product concentration= fraction of fluorescence increase*concentration of substrate.
From your time of the experiment, now velocity can be calculated.
To get the fluorescence intensity of the substrate when it is fully converted to product, you can either have the 2 products synthesized (costly, but a really good idea), or you can use a high enough enzyme concentration to convert all of the substrate to product.
A complication arises if the substrate concentration is high enough that the inner filter effect becomes significant. The inner filter effect is a reduction in fluorescence intensity caused by absorption of light by the sample itself. This is generally insignificant at nanomolar concentrations, but can become significant at micromolar concentrations. It will be detected as a sub-linear increase in fluorescence intensity as a function of concentration.
Mr. Seo,
To quantify Fs intensity means to quantify the integral intensity or the area under the Fs pattern. The error contribution to the intensity value is significant. Thus the error contribution to any kinetic or thermodynamic parameter is significant. In other words, the FRET assay does not provide reliable analytical information from the perspective of the chemometrics, which directly produces a nonreliable kinetic data. Also, Fs method itself is characterized by high method performances such as LODs and LOQs.
Conversely, the method of choice for quantification of kinetic and thermodynamic data, including diffusion one; in addition to, 3D structural determination of the analytes in vivo is: Mass spectrometry.
Quantification of kinetics can be carried out by our innovative formulas connecting experimental measurable variable MS intensity with the kinetic rate 'k' shown in [1]. Our equations are applicable despite the ionization MS method.
[1] Article Quantitative collision induced mass spectrometry of substitu...
Quantification of diffusion parameter (DSD) and determination of the analyte concentration at pg.(mL)-1 range at a reliability in chemometric terms r = 1 (or exact or absolute quantification,) can be carried out, again, using our model formulas [2a-c].
[2] (a) Article Stochastic dynamic electrospray ionization mass spectrometri...
[2] (b) Article A mass spectrometric stochastic dynamic diffusion approach t...
[2] (c) Article Electrospray ionization mass spectrometric solvate cluster a...
Both the kinetic and diffusion parameters are connected mutually. In addition, they are connected with thermodynamic ones.
Consider the following useful discussions [3a-c] and the reference section therein.
[3] (a) https://www.researchgate.net/post/any_expert_in_endotoxin_quantification_by_fluorescent_assay
[3] (b) https://www.researchgate.net/post/Anti-SARS-COV-2_Cytoprotection_Assay
[3] (c) https://www.researchgate.net/post/How_to_use_Sephadex_Column_to_isolate_aminoglycoside_compounds
Detail reading of the references provided shows that any assay based on Fs analysis does not provide meaningful analytical information due to a set of disadvantages of the Fs method. On the contrary, meaningful analytical information currently can be carried out only my mass spectrometry. The method of mass spectrometry is currently the method of choice for such analyses because of it shows superior instrumental characteristics, which are far beyond the capability of any other analytical instrumentation.
Therefore, quantification by Fs is highly obstructive from the perspective of the analytical science and the chemometrics or the statistical analysis. Such type studies are on the boundery of the speculation in the science from the perspective of the analytical science. The quantitative analysis, however, is part of the analytical sciences, in particular - analytical chemistry.
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