Abstract
For a complete understanding of biochemical reactions, information on complex stoichiometry is essential. However, measuring stoichiometry is experimentally challenging. Our lab has developed a FRET-based assay to study protein complex stoichiometry in vitro. This assay, also known as Job plot, is set up as a continuous variation of the molar ratio between the two species, kept at constant total concentration. The FRET (Fluorescence Resonance Energy Transfer) between the two fluorescently-labeled proteins is measured and the stoichiometry is inferred from the sample with highest FRET signal. This approach allows us to assess complex stoichiometry in solution.
Highlights
[Background] Each biochemical reaction requires the interaction between two or more cellular components
For the interaction between large particles that lead to dramatic molecular weight changes, stoichiometry can be inferred by low-resolution structural analysis
These approaches include size-exclusion chromatography, multi-angle light scattering, analytical ultracentrifugation, which are techniques capable of providing accurate molecular weights of the particles. These methods require a considerable amount of material and are prone to error when small molecular weight changes are to be observed
Summary
A. Fluorescence labeling of the histone-binding protein 1. In a minimum reaction volume of 400 μl, mix 10 μM of histone-binding protein with 10 μM of Atto647N maleimide dye, in PD-10 buffer. 5. Protein function should always be validated after labeling with fluorescence dyes. In the example in Supplementary file 1: the Kd is ~1 nM, we keep the total protein concentration in each tube at 150 nM. Use the worksheet attached to prepare the stock solutions These are prepared at twice the protein concentration required in the well reactions. 3. Final well reactions are prepared by mixing 20 μl of Protein[1] solution with 20 μl of Protein[2] solution, leading to the desired final protein concentration, as they are diluted two-fold in this step.
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