Hi, I'm reading a article where they screened SERT mutants for enhanced thermostability using high-throughput ligand binding assay and by fluorescence-detection size exclusion chromatography. Can anyone explain how it works?
I think this method uses a fluorescent based detection through a tag such GFP or tyrosine to determine how stable proteins are under different temperatures.
So in your case comparing temperature sensitivity of you wild type protein to your mutants will tell you the effect the mutations have on the protein when a similar temperature is applying for all protein variants.
Or how stable your mutants are upon ligand binding relative to the wild type under a giving temperature.
This article might be useful -
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3441139/
All the best!
Having no idea what paper you are talking about, I can only wildly guess.SEC (aka gel filtration) separates molecules by Stokes-radius. Thus, either they were separating substrate-enzyme complex away from free substrate and measured the fluorescence of the bound substrate, or they used the intrinsic fluorescence of the protein (Trp) to measure the oligomerisation of the protein itself. The later can be an effect of temperature dependent inactivation, substrate binding can protect enzymes against denaturation. I have described such experiments in doi:10.1007/978-1-4419-7251-4_35.
High-throughput binding can be done by many methods, often surface plasmon resonance is used: The protein is bound at a surface, and the refractive index of the medium next to that surface is measured. If substrates bind to the protein, the refractive index is increased, dissociation leads to a decrease. Temperature-dependent denaturation would lead to changes in affinity. Chapter 38 of the same book deals with that sort of experiments.
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