How can you study protein-ligand interactions with fluorescence spectroscopy?

1. FRET of ligand receptor pair 2. Tryptophan fluorescence if the receptor conformational change is detectable by this method. 3. displacement of a low affinity fluorescent LMW binder that has increased fluorescence when buried in the ligand pocket. Ligand binding will decrease fluorescence. 4. surface LMW binder that increases fluorescence when buried by ligand binding that also decreases hydration of the LMW fluorescent binder. ligand binding will increase fluorescence.

Use a Co-IP by selecting an antibody that targets a known protein that is believed to be a member of a larger complex of proteins. By targeting this known member of a complex with an antibody, you might be able to pull the entire protein complex out of solution and thereby identify unknown members of the complex. The immune complex is then captured, or precipitated, on a beaded support to which an antibody-binding protein is immobilized (such as Protein A or G), and any proteins not precipitated on the beads are washed away. We can conjugate the antibody with a fluorophore to measure the degree of absorbante using Spector

One way is to use titration methode which compromises the emitted fluorescence by adding your protein, ligand or the competitor considering the availability and value of your reactants. Based on the nature of your reactants you could have a proper DOE which could include the type and size of the cuvette, initial volume, injection volume, time of the reaction and et. c.

For a quick approach and if antibodies are available, I would immediately consider a PLA experiment- in situ Proximity ligation assay. No fancy apparatus is needed, and it is the price of a kit. With a microscope and proper controls, one can directly evaluate if the protein can interact.

Firstly assess whether there is an intrinsic fluorescence change in the protein upon binding ligand. Perform a fluorescence spectroscopic analysis (i.e. emission spectrum) plus/minus the ligand (the latter should be at a high concentration). If you see a change in the emission spectrum, exploit that for your measurement of ligand binding. This is the simplest solution, if it doesn't show a change move onto the more complex approaches.

If starting completely from scratch, utilize the Florescent Protien Database FRET tool. With this tool you can select an optimal FRET pair for screening protien:protien interactions in vitro. The best par will generate the best yield while minimizing bleed. Examples include CyPet and YPet or novel pairs like LSSmGFP and mscarlet3.

My major concern is the distinct possibility of a fluorochrome label interfering with endogenous behaviors. I would recommend using a proximity assay with oligo-conjugated validated antibodies. Using a rolling amplification with fluorescent labeled dNTPs after ligation detects proximity to proteins and/or nucleic acids (use of labeled probes rather than antibody). You can multiplex using ddNTPs to terminate one reaction, restain for second target pair, and use a different color dNTP during amplification. Allows for simultaneous detection of multiple interactions with intracellular localization imaging available.

One tool that can be used is measuring fluorescence resonance energy transfer. This is based on Forster Resonance Energy Transfer principles. In this method, two biomolecules are labeled. One donor and one acceptor. If the molecules are close enough the donor will emit energy to the acceptor upon donor excitation. The strength of interaction can be measured as a function of fluorescent intensity. These experiments can be run either in living cells or in a test tube. I've used this technique extensively in my graduate career.

Fluorescence polarization and anisotropic are fantastic, easy, first-shot approaches! Just need to be weary that labeling a small molecule can change its binding properties.

The best advantage is competitive assays. With in vitro FRET based assays you can test for compounds that can separate a protein:protein interaction. Other methods can only provide single time point analysis as they often involve the addition of readout reagents (nanoBRET) or other wise damaging the interacting/denaturing partners.

You also need to consider non-specific interactions between the florescent protiens. If using a FRET based assay be sure that your selected florescent protiens are true monomers and do not naturally dimerze!

Here I want to add a demystified version of the explanation to be understood by everyone. Fluorescence spectroscopy resembles a mystery code that particles use to speak with researchers. It's a method for uncovering stowed-away insights concerning these minuscule structure blocks of life by making them light up in a flawlessly brilliant dance. How Can it Assist with concentrating on Protein-Ligand Communications? Proteins resemble the hosts of a party, and ligands are their unique visitors. Fluorescence spectroscopy permits us to perceive how these visitors (ligands) connect with the hosts (proteins). At the point when they tie together, they make an unmistakable gleam, uncovering their connection designs.