Biomolecular Interaction Detection Techniques

In this video, we describe the field-effect biosensing (FEB) technology to detect and quantify protein-protein interactions. The target proteins immobilized on the graphene biosensor interact with analyte proteins in solution, resulting in electrical current alterations in the FEB system. This change in the electrical response indicates the binding affinity between the proteins.

In this video, we demonstrate the use of mass photometry to study antigen-antibody interactions in a flow chamber. The focused light scatters differently based on the mass of the molecules. A detector collects the scattered light and generates the interference pattern as spots of varied intensities, which correlate with the molecular masses.

The video describes the FLIM-FRET imaging technique to determine the protein-protein interaction in live bacteria expressing cytoplasmic proteins labeled with fluorescent proteins, a donor eGFP, and acceptor mCherry. The combined technique also allows the quantification of the interacting proteins.

In this video, we demonstrate the technique to measure transcellular interactions between synaptic cell adhesion molecules through the HEK cell aggregation assay. In this assay, an equal number of HEK cells expressing a desired presynaptic cell adhesion molecule and a fluorescent protein are mixed with HEK cells expressing a post-synaptic cell adhesion molecule and another fluorescent protein. The interaction between the cell adhesion molecules forces the cells to aggregate, which can be...

This video describes the proximity ligation assay to study in situ protein-protein interactions in nematode cells. The close proximity of the proteins of interest generates the circular DNA template with the help of a PLA probe attached to a secondary antibody. The probe is further amplified using the amplification solution, which produces a long single-stranded DNA followed by the hybridization of the detector probe, enabling the localization of protein-protein interactions in cells.

This video demonstrates co-immunoprecipitated protein complexes on a PVDF membrane for protein-protein interaction analysis. The reduced proteins from complexes were treated with trypsin to cleave the individual proteins at the smaller peptides, then, the remaining peptides were extracted from the PVDF membrane. The pulled peptide derived from both proteins in the complex was then dried and resuspended in a low concentration of formic acid for further analysis.

In this video, we demonstrate biolayer interferometry (BLI) technology to detect the intermolecular interactions between ligands and target analytes in real-time. BLI measures changes in the interference pattern of white light reflected off a layer of immobilized ligands on a biosensor surface as they interact with analytes in solution.

This video demonstrates the use of fluorescence fluctuation spectroscopy to detect the interaction among cell surface proteins at cell-cell contacts. By expressing the transmembrane adhesion receptor of interest labeled with a fluorescent protein and mixing two different cell populations harboring two spectrally separated fluorescent labels, the trans-interaction between the receptors of two neighboring cells with different-colored fluorescence is assessed via cross-correlation in the...

In this video, we demonstrate a technique to perform the co-immunoprecipitation of GFP-tagged wild-type and mutated PD-1 proteins using beads coated with anti-GFP antibodies. The bead-bound PD-1-GFP proteins are incubated with SHP2 proteins that interact with PD-1, enabling the identification of specific structural motifs in PD-1 that play a critical role in SHP2 interaction.

This video describes an ELISA-based technique that detects protein-protein interactions between two proteins by measuring the binding of the second protein to the immobilized first protein. The bound second protein is detected using specific antibodies labeled with an enzyme, such as horseradish peroxidase.

This video demonstrates a conductimetric biosensor assay to study the interaction of an antibody with a target antigen immobilized on a transducer chip. The biosensor is engineered to have a bifunctional chimeric protein — a β-lactamase enzyme with a functional catalytic site fused to a nanobody for binding to the immobilized target antigen. Upon binding, the catalytic hydrolysis of an antibiotic substrate by the β-lactamase enzyme generates an electrical signal — confirming the...

This video demonstrates a technique that uses biotinylated cell-penetrating peptides to detect intracellular protein-protein interactions. The cell-penetrating peptides facilitate the conjugated peptide to enter a cell and specifically bind to its interacting proteins. The biotin, after binding to avidin, allows purification of the interacting proteins’ complex from a cell lysate.

This video demonstrates the yeast two-hybrid assay to detect specific protein self-interactions. In this assay, the protein of interest is tagged to two domains of a transcription factor that encodes the β-galactosidase enzyme. When the two proteins interact, this causes transcription and translation to form the enzyme, which is then detected using a biochemical assay.

This video demonstrates tandem affinity purification — a technique to purify protein complexes from eukaryotic cells to study protein-protein interaction. The complex contains two proteins labeled with different epitope tags. Upon the purification of the complex using two resins with an affinity for the two different tags in a sequential manner, the presence of both proteins in the elute confirms the interaction between the two.

This video describes the surface plasmon resonance, or SPR, technique for studying the interaction between two molecules using a sensor chip. The change in resonance angle and the intensity of the reflected light are detected as a function of the association and dissociation of two molecules, and this provides information about the interactions between the two molecules.

This video describes the nuclear magnetic resonance spectroscopy technique to study protein-protein interactions between 15N-labeled wild-type and mutant envoplakin proteins and the unlabeled vimentin protein. The successful interaction between wild-type envoplakin and vimentin leads to extensive line broadening and peak disappearance in the NMR spectra, whereas the absence of an interaction between the mutated envoplakin and vimentin results in well-resolved peaks in the NMR spectra.

In this video, we demonstrate the phage display technique to isolate pre-engineered phages expressing proteins with high affinity to the target protein.

This video demonstrates the technique of fluorescence fluctuation spectroscopy (FFS) to study protein homo-oligomerization. Fluorescently-labeled proteins in a sample are dimerized using an agent when studying protein oligomerization using FFS. Using a confocal microscope, as the proteins move in and out of the small observation volume, the analysis of the fluctuations in the brightness of the fluorescent molecules is performed to determine the oligomeric state of the proteins.

This video describes BiFC-PALM ― a combination of photoactivated localization microscopy and bimolecular fluorescence complementation ― to assess protein-protein interactions. BiFC involves the fusion of two fluorescent protein fragments with two interacting proteins of interest. When the two proteins interact, the fragments are brought into proximity, which allows the two parts to come together and reconstitute a functional fluorescent protein. The fluorescence of a single fluorophore is...

In this video, we describe the fluorescence anisotropy technique to study the interactions between the fluorophore-tagged Shwachman-diamond syndrome (SBDS) protein and the elongation factor-like 1 GTPase (EFL1). On incubating SBDS proteins with gradually increasing concentrations of EFL1, a steady increase in anisotropy is observed, indicating a successful interaction between the two proteins.

In this video, we demonstrate the in-cell nuclear magnetic resonance spectroscopy technique to study protein-ligand interactions between unlabeled overexpressed proteins and small molecules. The successful ligand-protein interaction is confirmed visually by the appearance of an additional set of peaks in the spectral region of interest that gradually replaces the original peaks.

This video demonstrates a method to study protein-drug interactions using QCM biosensor-based biopanning of the T7 phage-displayed peptide library. The biopanning of phages displaying a specific peptide on the QCM biosensor allows specific isolation of high-affinity peptides, followed by amino acid sequencing, which helps to identify specific interaction sites on the target protein.

This video describes SEC-MALS, which leverages the abilities of size-exclusion chromatography (SEC) and multi-angle light scattering (MALS). In SEC-MALS, the SEC separates proteins or protein complexes based on their size, and the MALS detector determines the average molecular weight by measuring the intensity of the light scattered by the molecule at different angles.

In this video, we have demonstrated bimolecular complementation affinity purification, BiCAP, to detect and isolate specific protein dimers using conformation-specific single-domain antibodies.

In this video, we describe a flow cytometry-based competitive binding assay to detect the interactions between the CXC Chemokine Receptor 4 (CXCR4) and its fluorescently-labeled natural ligand CXC Chemokine Ligand 12 (CXCL12), in the presence of a CXCR4-targeting small molecule. Incubating CXCR4-expressing cells with lower small molecule concentrations allows CXCR4-CXCL12 binding to some extent, which progressively declines upon a gradual increase in small molecule concentrations.

This video demonstrates the reporter assay-based detection of the receptor-ligand interaction. The successful receptor-ligand interaction activates the downstream signaling pathway, causing the generation and release of cytokines as secretory reporter molecules in the solution, which is confirmed using ELISA.

In this video, biomembrane force probes (BFPs) are used to measure the forces between receptors and ligands. This technique can detect interactions by monitoring the pressure needed to break the bonds between the pMHC conjugated on the probe beads and the TCRs on the target cells.

This video demonstrates a competition assay to study GTPase-binding protein partners. Utilizing nucleotide-bound GTPase protein immobilized on magnetic beads, the competitive binding between two interacting protein partners for the same binding site on the GTPase can be studied to assess the binding affinities of the protein partners.

In this video, we demonstrate the process for detecting antibody isotypes in multiple human serum samples using a microarray slide printed with antigenic subtypes of influenza virus strains.

In this video, we demonstrate the electrophoretic mobility shift assay to detect protein-DNA interactions using infrared fluorescent-labeled DNA probes. When the protein binds to the DNA probe, it forms a large complex that migrates slowly on and appears as a shifted band on the gel.

In this video, we demonstrate the chromatin immunoprecipitation technique to identify protein binding sites on specific regions of the genomic DNA of oligodendrocyte precursor cells via the selective immunoprecipitation of protein-bound chromatin fragments. This method helps to study the interaction of several regulatory proteins, including transcription factors involved in gene regulation.

This video describes high-performance fluorescence anisotropy that helps to monitor the interaction between transcription factors and DNA. The assay monitors the interactions of a fluorophore-labeled DNA molecule with its specific transcription factor by measuring the degree of polarization due to molecular rotation or anisotropy of the fluorophore-labeled DNA.

This video demonstrates an in vivo assay in bacteria to study the interaction of RNA-binding proteins (RBPs) with RNA. The bacterial cells are transformed with two plasmid constructs — a binding-site plasmid encoding an mRNA containing a fluorescent reporter gene downstream of an RBP-binding site — while an RBP plasmid expresses the RBP proteins under the control of an inducer. Upon inducer-mediated increase in RBP production, the RBPs bind to the mRNA, resulting in translational repression of...

In this video, we demonstrate the modified yeast one-hybrid assay, which detects multiple proteins interacting with target DNA. The transformed yeast cells contain the target DNA sequence upstream of the reporter gene. Additionally, they express transcription factor proteins. When the proteins form complexes and bind to the target DNA, the reporter gene is expressed and can be detected enzymatically.

In this video, we demonstrate the procedure to determine the helicase activity of a target protein to unwind the biotin-labeled dsRNA substrate. The activity of the enzyme was identified by analyzing the electrophoretic mobility shift, followed by a chemiluminescence assay using chemiluminescent enzyme-conjugated streptavidin.

In this video, we describe the immunofluorescence method to visualize the localization of the DNA repair proteins γH2AX and 53BP1 at the sites of double-strand DNA breaks in irradiated cell nuclei.

This video demonstrates an in vitro RNA pull-down assay to identify RNA-binding proteins (RBPs), which interact with the adenylate-uridylate-rich element (ARE) sequences in mRNA. The target RBPs from a cell lysate are mixed with an RNA probe to form RNA-protein complexes. The complexes are isolated via affinity purification utilizing the affinity of the desthiobiotin label of the RNA probe to a streptavidin-labeled magnetic bead.

In this video, we describe the systematic evolution of ligands by the exponential enrichment (SELEX) method to identify specific RNA-binding sequences for a target protein of interest. The protein is incubated with a large pool of randomized RNA sequences, and the protein-binding RNA sequences are isolated, PCR-amplified, and sequenced to identify their protein-binding sites.

This video describes a method to visualize the assembly of fusion protein condensates on DNA molecules at specific loci using single-molecule imaging. These condensates are formed due to LCD-LCD and LCD-DBD interactions, which allow for the recruitment of RNA polymerase II and enhanced gene transcription.

This video demonstrates differential scanning calorimetry — a technique to study the interaction between a biomolecule and a ligand. The binding of a DNA aptamer and a thermolabile ligand is studied by applying repeated heating and cooling cycles to the sample and obtaining scans for thermodynamic analysis.

This video demonstrates microscale thermophoresis for studying the protein-lipid interaction. The assay detects the interaction between molecules by quantifying the thermophoretic movement of fluorescent-labeled proteins in response to a temperature gradient. The fluorescent molecule is mixed with different concentrations of the non-fluorescent lipid molecules, and the mixture of molecules in the solution is loaded into capillaries. A temperature gradient is applied to the samples in the...

In this video, we demonstrate the interaction between proteins and artificial phospholipid vesicles using quantitative flow cytometry. The binding of fluorescently-labeled proteins to fluorescently-labeled phospholipid vesicles increases the mean fluorescence intensity, and this increase is detected by a flow cytometer.

This video demonstrates a dissipative microgravimetry technique to investigate the binding of proteins to lipid bilayers. Small unilamellar vesicles are added to coat the surface of the quartz sensor of a microbalance. Utilizing the calcium ion-dependent binding of the target phospholipid-binding protein to the bilayer, changes in the oscillation frequency of the sensor and the dissipation of the oscillation are monitored to determine the nature of the protein-lipid bilayer binding.

In this video, we demonstrate protein-phosphoinositide interactions in a microfluidic pattern using the pH modulation assay, using pH-sensitive fluorescent dye tagged to phosphatidylethanolamine lipid of a supported lipid bilayer, called a microfluidic platform. The modulations in pH after protein binding to phospholipid cause fluorescence quenching.