1. ATPase assay:
ATPase assays measures the rate of ATP hydrolysis into inorganic phosphate and ADP. Released inorganic phosphate is measured more commonly through either absorbance or radioactivity based assays. In absorbance based methods, the released inorganic phosphate is detected calorimetrically by its binding to Malachite Green dye. Malachite Green, molybdate and free phosphate forms a green colored complex which shows absorbance at 620nm. In radioactivity based assay, labelled ATP (P32-γ) is used as substrate that gets hydrolyzed to release inorganic labelled phosphate (P32-γ) which is quantified in a scintillation counter.
2. Substrate refolding assays:
Substrate refolding assays are the functional assays to calculate the protein refolding activity of chaperones. Firefly Luciferase, Malate Dehydrogenase, Lactate Dehydrogenase are widely used substrates to monitor chaperoning activity. The substrates are denatured either thermally or chemically, and refolded in the presence and absence of desired chaperones. The refolding is measured by increase in the native activity of the substrate as it refolds as a function of time.
3. Surface plasmon resonance :
Surface Plasmon Resonance (SPR) is widely used to measure the kinetics and equilibrium constants of protein-protein or protein-small molecules interactions. For SPR studies, one of binding partner (the ligand) is immobilized on the sensor surface, and the interaction is studied by monitoring increase in response unit as the analyte is flown over the surface. For equilibrium measurements, the analyte is flown at different concentrations over the ligand bound sensor surface. The response unit monitored over a period of time generates sensorgram. The fit of the sensorgram datato appropriate binding model provides binding rate constants and equilibrium binding affinities of interacting molecules.
4. Isothermal titration calorimetry:
Isothermal titration calorimetry (ITC) provides thermodynamical parameters of a binding event by measuring change in heat of the reaction.The solution containing analyte is filled in sample cell and titrated by adding series of small volume of ligand. During interaction between the molecules, heat is either released or absorbed which is accurately measured in ITC. As more and more ligand is added, analyte gets more and more saturated with the ligand, and thus less of heat gets transferred. The area of each peak corresponding to the ligand injections is calculated and plotted against the molar ratio of ligand to analyte. ITC measures thermodynamic parameters of binding molecules in solution and does not require labelling or immobilization of any of the interacting partner. Thermodynamic parameters such as Enthalpy change (∆H), Gibbs free energy changes (∆G) and Entropy changes (∆S) can be determined using ITC.
5. Fluorescence polarization assay :
The fluorescent polarization is used for quantitative estimation of protein-protein, protein-small molecule or protein-nucleic acid interactions. The assay is based upon the principle that fluorescence polarization obtained upon excitation of fluorescently labelled molecule by a plane polarized light is inversely proportional to the rate of molecular rotation in solution. As interaction between molecules forms a larger molecular weight complex with reduced rotational speed, there is change in degree of fluorescence polarization. The emission light intensity parallel and perpendicular to the excitation plane is recorded, and the difference between the two is normalized with respect to total fluorescence emission intensity to obtain fluorescence polarization.