There are many methods to investigate protein–protein interactions which are the physical contacts of high specificity established between two or more protein molecules involving electrostatic forces and hydrophobic effects.
Co-immunoprecipitation is considered[citation needed] to be the gold standard assay for protein–protein interactions, especially when it is performed with endogenous (not overexpressed and not tagged) proteins.
Pull-down assays are a common variation of immunoprecipitation and immunoelectrophoresis and are used identically, although this approach is more amenable to an initial screen for interacting proteins.
Upon exposure to ultraviolet light, the diazirines are activated and bind to interacting proteins that are within a few angstroms of the photo-reactive amino acid analog.
[6] Tandem affinity purification (TAP) method allows high throughput identification of protein interactions.
In contrast to yeast two-hybrid approach the accuracy of the method can be compared to those of small-scale experiments[7] and the interactions are detected within the correct cellular environment as by co-immunoprecipitation.
However, the TAP tag method requires two successive steps of protein purification and consequently it can not readily detect transient protein–protein interactions.
After the amplification reaction, several-hundredfold replication of the DNA circle has occurred and flurophore or enzyme labeled complementary oligonucleotide probes highlight the product.
[citation needed] SPR instruments measure the change in the refractive index of light reflected from a metal surface (the "biosensor").
This insensitivity permits DLS measurements from 1 μL volumes in 1536 well plates, and lowers sample requirements into the femtomole range.
Flow-induced dispersion analysis (FIDA), is a new capillary-based and immobilization-free technology used for characterization and quantification of biomolecular interaction and protein concentration under native conditions.
[17] The technique is based on measuring the change in apparent size (hydrodynamic radius) of a selective ligand when interacting with the analyte of interest.
A FIDA assay works in complex solutions (e.g. plasma [18]), and provides information regarding analyte concentration, affinity constants, molecular size and binding kinetics.
Furthermore, the two-photon and three-photon excitation practically eliminates photobleaching effects and provide ultra-fast recording of FCCS or FCS data.
[19][20][21] [22] Applied in vivo, FRET has been used to detect the location and interactions of genes and cellular structures including integrins and membrane proteins.
[29] Isothermal titration calorimetry (ITC), is considered as the most quantitative technique available for measuring the thermodynamic properties of protein–protein interactions and is becoming a necessary tool for protein–protein complex structural studies.
ITC provides information regarding the stoichiometry, enthalpy, entropy, and binding kinetics between two interacting proteins.
[30] Microscale thermophoresis (MST), is a new method that enables the quantitative analysis of molecular interactions in solution at the microliter scale.
MST provides information regarding the binding affinity, stoichiometry, competition and enthalpy of two or more interacting proteins.
[31][32] Rotating cell‑based ligand binding assay using radioactivity or fluorescence, is a recent method that measures molecular interactions in living cells in real-time.
[37] In this technology, the living or fixed cells are physically retained on the surface of biosensor chips using biocompatible and flow-permeable polymer traps.
Single colour reflectometry (SCORE) is a label-free technology for measuring all kinds of biomolecular interactions in real-time.
Upon binding of an analyte to the ligand, the real-time kinetic rates (kon, koff) can be measured as changes in fluorescence intensity and the Kd can be derived.
[40] switchSENSE can additionally be utilized to detect conformational changes induced by ligands binding to a target protein.
The methods in this section are primarily computational although they typically require data generated by wet lab experiments.