Overview of Protein–Protein Interactions

PPIs occur when two or more proteins physically interact, driving nearly all biological processes. Examples include enzyme-substrate interactions, receptor-ligand signaling cascades, cell cycle regulation, and structural assemblies like the cytoskeleton.

Yes, proteins interact with other proteins. These interactions are crucial for many biological processes, including signal transduction, cellular structure, and metabolic pathways.

Breaking protein-protein interactions can be achieved through several methods:

• Small Molecules: Designing or using small molecules that can bind to one of the proteins and disrupt the interaction.
• Peptides: Using peptides that mimic the interaction site to competitively inhibit the protein-protein interaction.
• Mutagenesis: Altering amino acids at the interaction site through genetic engineering to prevent binding.
• Antibodies: Antibodies that specifically bind to one of the proteins block the interaction.
• Environmental Changes: Altering conditions such as pH, temperature, or ionic strength to destabilize the interaction. This is often used to regenerate biosensor to re-use them in binding assays.
• Protein Degradation: Using techniques like targeted protein degradation to remove one of the interacting proteins.

These methods are often used in research and drug development to study protein functions and develop therapeutic interventions.

Testing for PPIs involves several steps and can be achieved using various techniques. 

First, choose the appropriate method based on the nature of the proteins, the type of interaction, and the information needed, such as binding affinity, kinetics, or structural details.

Next, prepare protein samples. If required, label the proteins with fluorescent or other suitable tags to facilitate detection, especially for techniques like FRET or MST.

After conducting the experiment depending on the chosen method, analyze the data. Finally, validate the results by confirming findings using complementary techniques or repeating experiments to ensure reliability and reproducibility.

Common techniques used to study protein-protein interactions (PPI) include:

• Co-immunoprecipitation (Co-IP): A method to detect physical interactions between proteins using specific antibodies.
• Yeast Two-Hybrid (Y2H) 선별: A genetic approach to identify protein interactions by detecting the activation of 정보제공 유전자 in yeast.
• Biolayer Interferometry (BLI): A label-free technique for measuring binding kinetics, affinity and analyte concentration in real-time without the need for microfluidics.
• 표면 플라스몬 공명 (SPR): Another label-free technique to measure binding kinetics and affinity in real-time.
• X-ray Crystallography: Used to determine the 3D structure of protein complexes at atomic resolution.
• Nuclear Magnetic Resonance (NMR) Spectroscopy: Provides information on the structure and dynamics of protein complexes in solution.
• Fluorescence Resonance Energy Transfer (FRET): A technique to study interactions by measuring energy transfer between two fluorescently labeled proteins.
• Mass Spectrometry (MS): Used to identify and quantify proteins in complexes, often combined with cross-linking or affinity purification.
• Isothermal 적정 Calorimetry (ITC): Measures the heat change during protein binding to determine interaction thermodynamics.
• MicroScale Thermophoresis (MST): Measures the movement of molecules in temperature gradients to study binding affinities and kinetics.

Biolayer Interferometry (BLI) is a technique used to study protein-protein interactions. It measures binding events in real-time by detecting changes in the interference pattern of light reflected from a biosensor surface. One protein is immobilized on the sensor, and the other is in solution. BLI provides information on binding kinetics, affinity, and specificity without the need for labeling, making it a valuable tool for analyzing protein interactions. 

The BLI technology allows a fluidic-free design. Unlike other techniques that require complex microfluidic systems, BLI uses a simple dip-and-read format eliminating the risk of clogging and cross-contamination and simplifying the experimental setup.

Additionally, BLI is capable of measuring interactions in crude samples, such as cell lysates or unpurified protein mixtures. This capability is particularly beneficial for early-stage research and high-throughput 선별.

For Biolayer Interferometry (BLI) experiments, the amount of protein required can vary depending on the specific assay and the proteins involved. BLI utilizes a plate-based format, which allows for non-destructive measurement. This means that the sample plate can be reused for further experiments after the initial BLI analysis. This feature not only conserves valuable samples but also enables subsequent analyses or assays, enhancing the efficiency and versatility of the experimental workflow. It's important to optimize the concentration and amount based on preliminary experiments to ensure reliable and reproducible results.

Generally, for the experiment you need:

• Immobilized Protein: Typically, a few micrograms (µg) of the protein are needed to immobilize on the biosensor. The exact amount depends on the protein's molecular weight and the immobilization strategy.
• Analyte Protein: For the protein in solution (analyte), concentrations in the range of nanomolar (nM) to micromolar (µM) are commonly used.

표면 플라스몬 공명 (SPR) is a technique used to study protein-protein interactions. It allows researchers to measure the binding affinity, kinetics, and specificity of interactions between proteins in real-time without the need for labeling. In SPR, one protein is immobilized on a sensor chip, and the other protein is flowed over the surface. Changes in the refractive index near the surface are measured, which occur when the proteins interact. This provides valuable data on how proteins interact, including the strength and duration of the interaction.

The amount of protein required for 표면 플라스몬 공명 (SPR) experiments can vary depending on the specific setup and the proteins involved. Generally, you need:

• Immobilized Protein: Typically, a few micrograms (µg) of the protein are needed to immobilize on the sensor chip. The exact amount depends on the protein's molecular weight and the immobilization method.
• Analyte Protein: For the protein in solution (analyte), concentrations in the range of nanomolar (nM) to micromolar (µM) are commonly used. The total volume required can range from a few hundred microliters (µL) to a few milliliters (mL), depending on the experimental design and the number of replicates.

It's important to optimize the concentration and amount based on preliminary experiments to ensure reliable and reproducible results.