There are many methods to investigate
protein–protein interaction
Protein–protein interactions (PPIs) are physical contacts of high specificity established between two or more protein molecules as a result of biochemical events steered by interactions that include electrostatic forces, hydrogen bonding and th ...
s which are the physical contacts of high specificity established between two or more protein molecules involving
electrostatic force
Coulomb's inverse-square law, or simply Coulomb's law, is an experimental law of physics that quantifies the amount of force between two stationary, electrically charged particles. The electric force between charged bodies at rest is conventio ...
s and
hydrophobic effect
The hydrophobic effect is the observed tendency of nonpolar substances to aggregate in an aqueous solution and exclude water molecules. The word hydrophobic literally means "water-fearing", and it describes the segregation of water and nonpola ...
s. Each of the approaches has its own strengths and weaknesses, especially with regard to the
sensitivity and specificity
In medicine and statistics, sensitivity and specificity mathematically describe the accuracy of a test that reports the presence or absence of a medical condition. If individuals who have the condition are considered "positive" and those who do ...
of the method. A high sensitivity means that many of the interactions that occur are detected by the screen. A high specificity indicates that most of the interactions detected by the screen are occurring in reality.
Biochemical methods
Co-immunoprecipitation
Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sam ...
is considered to be the gold standard assay for protein–protein interactions, especially when it is performed with endogenous (not overexpressed and not
tagged) proteins. The protein of interest is isolated with a specific
antibody
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and Viral disease, viruses. The antibody recognizes a unique m ...
. Interaction partners which stick to this protein are subsequently identified by
Western blotting
The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecti ...
. Interactions detected by this approach are considered to be real. However, this method can only verify interactions between suspected interaction partners. Thus, it is not a screening approach. A note of caution also is that immunoprecipitation experiments reveal direct and indirect interactions. Thus, positive results may indicate that two proteins interact directly or may interact via one or more bridging molecules. This could include bridging proteins, nucleic acids (DNA or RNA), or other molecules.
Bimolecular fluorescence complementation (BiFC) is a new technique in observing the interactions of proteins. Combining with other new techniques, this method can be used to screen protein–protein interactions and their modulators,
DERB.
Affinity electrophoresis
Affinity electrophoresis is a general name for many analytical methods used in biochemistry and biotechnology. Both qualitative and quantitative information may be obtained through affinity electrophoresis. The methods include the so-called elect ...
as used for estimation of
binding constant
The binding constant, or affinity constant/association constant, is a special case of the equilibrium constant ''K'', and is the inverse of the dissociation constant. It is associated with the binding and unbinding reaction of receptor (R) and lig ...
s, as for instance in
lectin
Lectins are carbohydrate-binding proteins that are highly specific for sugar groups that are part of other molecules, so cause agglutination of particular cells or precipitation of glycoconjugates and polysaccharides. Lectins have a role in rec ...
affinity electrophoresis or characterization of molecules with specific features like
glycan
The terms glycans and polysaccharides are defined by IUPAC as synonyms meaning "compounds consisting of a large number of monosaccharides linked glycosidically". However, in practice the term glycan may also be used to refer to the carbohydrate ...
content or
ligand
In coordination chemistry, a ligand is an ion or molecule ( functional group) that binds to a central metal atom to form a coordination complex. The bonding with the metal generally involves formal donation of one or more of the ligand's ele ...
binding.
Pull-down assays are a common variation of
immunoprecipitation
Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sam ...
and
immunoelectrophoresis
Immunoelectrophoresis is a general name for a number of biochemical methods for separation and characterization of proteins based on electrophoresis and reaction with antibodies. All variants of immunoelectrophoresis require immunoglobulins, al ...
and are used identically, although this approach is more amenable to an initial screen for interacting proteins.
Label transfer can be used for screening or confirmation of protein interactions and can provide information about the interface where the interaction takes place. Label transfer can also detect weak or transient interactions that are difficult to capture using other ''in vitro'' detection strategies. In a label transfer reaction, a known protein is tagged with a detectable label. The label is then passed to an interacting protein, which can then be identified by the presence of the label.
Phage display
Phage display is a laboratory technique for the study of protein–protein, protein–peptide, and protein– DNA interactions that uses bacteriophages (viruses that infect bacteria) to connect proteins with the genetic information that encodes ...
is used for the high-throughput screening of protein interactions.
''In-vivo'' crosslinking of protein complexes using
photo-reactive amino acid analog Photo-reactive amino acid analogs are artificial analogs of natural amino acids that can be used for crosslinking of protein complexes. Photo-reactive amino acid analogs may be incorporated into proteins and peptides ''in vivo'' or in ''vitro''. Pho ...
s was introduced in 2005 by researchers from the Max Planck Institute In this method, cells are grown with
photoreactive diazirine
Diazirines are a class of organic molecules consisting of a carbon bound to two nitrogen atoms, which are double-bonded to each other, forming a cyclopropene-like ring, 3''H''-diazirene. They are isomeric with diazocarbon groups, and like the ...
analogs to
leucine
Leucine (symbol Leu or L) is an essential amino acid that is used in the biosynthesis of proteins. Leucine is an α-amino acid, meaning it contains an α- amino group (which is in the protonated −NH3+ form under biological conditions), an α- ...
and
methionine, which are incorporated into proteins. Upon exposure to ultraviolet light, the diazirines are activated and bind to interacting proteins that are within a few
angstrom
The angstromEntry "angstrom" in the Oxford online dictionary. Retrieved on 2019-03-02 from https://en.oxforddictionaries.com/definition/angstrom.Entry "angstrom" in the Merriam-Webster online dictionary. Retrieved on 2019-03-02 from https://www.m ...
s of the photo-reactive amino acid analog.
Tandem affinity purification Tandem affinity purification (TAP) is an immunoprecipitation-based purification technique for studying protein–protein interactions. The goal is to extract from a cell only the protein of interest, in complex with any other proteins it interacted ...
(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
and the interactions are detected within the correct cellular environment as by
co-immunoprecipitation
Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sam ...
. However, the TAP tag method requires two successive steps of protein purification and consequently it can not readily detect transient protein–protein interactions. Recent genome-wide TAP experiments were performed by Krogan ''et al.'' and Gavin ''et al.'' providing updated protein interaction data for yeast organism.
Chemical cross-linking
In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural ...
is often used to "fix" protein interactions in place before trying to isolate/identify interacting proteins. Common crosslinkers for this application include the non-cleavable
NHS
The National Health Service (NHS) is the umbrella term for the publicly funded healthcare systems of the United Kingdom (UK). Since 1948, they have been funded out of general taxation. There are three systems which are referred to using the " ...
-ester cross-linker,
bissulfosuccinimidyl suberate (BS3); a cleavable version of BS3,
dithiobis(sulfosuccinimidyl propionate) (DTSSP); and the
imidoester cross-linker
dimethyl dithiobispropionimidate (DTBP) that is popular for fixing interactions in
ChIP Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genom ...
assays.
Chemical cross-linking
In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural ...
followed by high mass
MALDI
In mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) is an ionization technique that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation. It has been applied to the analysis of ...
mass spectrometry
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a '' mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is u ...
can be used to analyze intact protein interactions in place before trying to isolate/identify interacting proteins. This method detects interactions among non-tagged proteins and is available from
CovalX.
SPINE (Strepprotein interaction experiment) uses a combination of reversible crosslinking with formaldehyde and an incorporation of an affinity tag to detect interaction partners ''in vivo''.
Quantitative immunoprecipitation combined with knock-down (QUICK) relies on co-immunoprecipitation, quantitative
mass spectrometry
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a '' mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is u ...
(
SILAC) and
RNA interference
RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression. Historically, RNAi was known by o ...
(RNAi). This method detects interactions among endogenous non-tagged proteins.
Thus, it has the same high confidence as co-immunoprecipitation. However, this method also depends on the availability of suitable antibodies.
Proximity ligation assay
Proximity ligation assay (in situ PLA) is a technology that extends the capabilities of traditional immunoassays to include direct detection of proteins, protein interactions, Extracellular vesicles and post translational modifications with high s ...
(PLA) in situ is an immunohistochemical method utilizing so called PLA probes for detection of proteins, protein interactions and modifications. Each PLA probes comes with a unique short DNA strand attached to it and bind either to species specific primary antibodies or consist of directly DNA-labeled primary antibodies. When the PLA probes are in close proximity, the DNA strands can interact through a subsequent addition of two other circle-forming DNA oligonucleotides. After joining of the two added oligonucleotides by enzymatic ligation, they are amplified via rolling circle amplification using a polymerase. After the amplification reaction, several-hundredfold replication of the DNA circle has occurred and flurophore or enzyme labeled complementary oligonucleotide probes highlight the product. The resulting high concentration of fluorescence or cromogenic signal in each single-molecule amplification product is easily visible as a distinct bright spot when viewed with either in a fluorescence microscope or a standard brightfield microscope.
Biophysical and theoretical methods
Surface plasmon resonance
Surface plasmon resonance (SPR) is the resonant oscillation of conduction electrons at the interface between negative and positive permittivity material in a particle stimulated by incident light. SPR is the basis of many standard tools for measu ...
(SPR) is the most common label-free technique for the measurement of biomolecular interactions. SPR instruments measure the change in the refractive index of light reflected from a metal surface (the "biosensor"). Binding of biomolecules to the other side of this surface leads to a change in the refractive index which is proportional to the mass added to the sensor surface. In a typical application, one binding partner (the "ligand", often a protein) is immobilized on the biosensor and a solution with potential binding partners (the "analyte") is channelled over this surface. The build-up of analyte over time allows to quantify on rates (kon), off rates (koff),
dissociation constants (Kd) and, in some applications, active concentrations of the analyte. Several different vendors offer SPR-based devices. Best known are
Biacore instruments which were the first commercially available.
Dual polarisation interferometry
Dual-polarization interferometry (DPI) is an analytical technique that probes molecular layers adsorbed to the surface of a waveguide using the evanescent wave of a laser beam. It is used to measure the conformational change in proteins, or oth ...
(DPI) can be used to measure protein–protein interactions. DPI provides real-time, high-resolution measurements of molecular size, density and mass. While tagging is not necessary, one of the protein species must be immobilized on the surface of a waveguide. As well as kinetics and affinity,
conformational change
In biochemistry, a conformational change is a change in the shape of a macromolecule, often induced by environmental factors.
A macromolecule is usually flexible and dynamic. Its shape can change in response to changes in its environment or oth ...
s during interaction can also be quantified.
Static light scattering
Static light scattering is a technique in physical chemistry that measures the intensity of the scattered light to obtain the average molecular weight ''Mw'' of a macromolecule like a polymer or a protein in solution. Measurement of the scattering ...
(SLS) measures changes in the
Rayleigh scattering
Rayleigh scattering ( ), named after the 19th-century British physicist Lord Rayleigh (John William Strutt), is the predominantly elastic scattering of light or other electromagnetic radiation by particles much smaller than the wavelength of the ...
of protein complexes in solution and can characterize both weak and strong interactions without labeling or immobilization of the proteins or other biomacromolecule. The composition-gradient, multi-angle static light scattering (CG-MALS) measurement mixes a series of aliquots of different concentrations or compositions, measures the effect of the changes in light scattering as a result of the interaction, and fits the correlated light scattering changes with concentration to a series of association models in order to find the best-fit descriptor. Weak, non-specific interactions are typically characterized via the second
virial coefficient
Virial coefficients B_i appear as coefficients in the virial expansion of the pressure of a many-particle system in powers of the density, providing systematic corrections to the ideal gas law. They are characteristic of the interaction potential ...
. For specific binding, this type of analysis can determine the stoichiometry and equilibrium association constant(s) of one or more associated complexes, including challenging systems such as those that exhibit simultaneous homo- and hetero-association, multi-valent interactions and cooperativity.
Dynamic light scattering
Dynamic light scattering (DLS) is a technique in physics that can be used to determine the size distribution profile of small particles in suspension or polymers in solution. In the scope of DLS, temporal fluctuations are usually analyzed usin ...
(DLS), also known as quasielastic light scattering (QELS), or photon correlation spectroscopy, processes the time-dependent fluctuations in scattered light intensity to yield the hydrodynamic radius of particles in solution. The hydrodynamic radius is the radius of a solid sphere with the same translational diffusion coefficient as that measured for the sample particle. As proteins associate, the average hydrodynamic radius of the solution increases. Application of the Method of Continuous Variation, otherwise known as the
Job plot Within chemistry, a Job plot, otherwise known as the method of continuous variation or Job's method, is a method used in analytical chemistry to determine the stoichiometry of a binding event. The method is named after Paul Job and is also used in ...
, with the solution hydrodynamic radius as the observable, enables ''in vitro'' determination of K
d, complex stoichiometry, complex hydrodynamic radius, and the Δ''H''° and Δ''S''° of protein–protein interactions. This technique does not entail immobilization or labeling. Transient and weak interactions can be characterized. Relative to static light scattering, which is based upon the absolute intensity of scattered light, DLS is insensitive to background light from the walls of containing structures. This insensitivity permits DLS measurements from 1 µL volumes in 1536 well plates, and lowers sample requirements into the femtomole range. This technique is also suitable for screening of buffer components and/or small molecule inhibitors/effectors.
Flow-induced dispersion analysis (FIDA), is a new capillary-based and immobilization-free technology used for characterization and quantification of biomolecular interaction and
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respon ...
concentration under native conditions.
The technique is based on measuring the change in apparent size (hydrodynamic radius) of a selective
ligand
In coordination chemistry, a ligand is an ion or molecule ( functional group) that binds to a central metal atom to form a coordination complex. The bonding with the metal generally involves formal donation of one or more of the ligand's ele ...
when interacting with the analyte of interest. A FIDA assay works in complex solutions (e.g. plasma ), and provides information regarding analyte concentration, affinity constants, molecular size and binding kinetics. A single assay is typically completed in minutes and only requires a sample consumption of a few µl.
Fluorescence polarization/anisotropy can be used to measure protein–protein or protein–ligand interactions. Typically one binding partner is labeled with a fluorescence probe (although sometimes intrinsic protein fluorescence from tryptophan can be used) and the sample is excited with polarized light. The increase in the polarization of the fluorescence upon binding of the labeled protein to its binding partner can be used to calculate the binding affinity.
With
fluorescence correlation spectroscopy
Fluorescence correlation spectroscopy (FCS) is a statistical analysis, via time correlation, of stationary fluctuations of the fluorescence intensity. Its theoretical underpinning originated from L. Onsager's regression hypothesis. The analysis p ...
, one protein is labeled with a fluorescent dye and the other is left unlabeled. The two proteins are then mixed and the data outputs the fraction of the labeled protein that is unbound and bound to the other protein, allowing you to get a measure of K
D and binding affinity. You can also take time-course measurements to characterize binding kinetics. FCS also tells you the size of the formed complexes so you can measure the stoichiometry of binding. A more powerful methods is
fluorescence cross-correlation spectroscopy (FCCS) that employs double labeling techniques and cross-correlation resulting in vastly improved signal-to-noise ratios over FCS. Furthermore, the two-photon and three-photon excitation practically eliminates photobleaching effects and provide ultra-fast recording of FCCS or FCS data.
Fluorescence resonance energy transfer
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...
(FRET) is a common technique when observing the interactions of different proteins.
Applied in vivo, FRET has been used to detect the location and interactions of genes and cellular structures including
integrins
Integrins are transmembrane receptors that facilitate cell-cell and cell-extracellular matrix (ECM) adhesion. Upon ligand binding, integrins activate signal transduction pathways that mediate cellular signals such as regulation of the cell cycle, ...
and membrane proteins. FRET can be used to obtain information about metabolic or signaling pathways.
Bio-layer interferometry (BLI) is a label-free technology for measuring biomolecular interactions (protein:protein or protein:small molecule). It is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time, providing detailed information regarding the kinetics of association and dissociation of the two molecule molecules as well as the affinity constant for the protein interaction (k
a, k
d and K
d). Due to sensor configuration, the technique is highly amenable to both purified and crude samples as well as high throughput screening experiments. The detection method can also be used to determine the molar concentration of analytes.
Protein activity determination by
NMR multi-nuclear relaxation measurements, or
2D-FT NMR spectroscopy in solutions, combined with nonlinear regression analysis of NMR relaxation or 2D-FT spectroscopy data sets. Whereas the concept of
water activity
Water activity (''aw'') is the partial pressure, partial vapor pressure of water in a solution divided by the standard state partial vapor pressure of water. In the field of food science, the standard state is most often defined as pure water a ...
is widely known and utilized in the applied biosciences, its complement—the
protein activity which quantitates protein–protein interactions—is much less familiar to bioscientists as it is more difficult to determine in dilute solutions of proteins; protein activity is also much harder to determine for concentrated protein solutions when protein aggregation, not merely transient protein association, is often the dominant process.
Isothermal titration calorimetry
Isothermal titration calorimetry (ITC) is a physical technique used to determine the thermodynamic parameters of interactions in solution. It is most often used to study the binding of small molecules (such as medicinal compounds) to larger macro ...
(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. This technique relies upon the accurate measurement of heat changes that follow the interaction of protein molecules in solution, without the need to label or immobilize the binding partners, since the absorption or production of heat is an intrinsic property of virtually all biochemical reactions. ITC provides information regarding the stoichiometry, enthalpy, entropy, and binding kinetics between two interacting proteins.
Microscale thermophoresis
Microscale thermophoresis (MST) is a technology for the biophysical analysis of interactions between biomolecules. Microscale thermophoresis is based on the detection of a temperature-induced change in fluorescence of a target as a function of the ...
(MST), is a new method that enables the quantitative analysis of molecular interactions in solution at the microliter scale. The technique is based on the thermophoresis of molecules, which provides information about molecule size, charge and hydration shell. Since at least one of these parameters is typically affected upon binding, the method can be used for the analysis of each kind of biomolecular interaction or modification. The method works equally well in standard buffers and biological liquids like blood or cell-lysate. It is a free solution method which does not need to immobilize the binding partners. MST provides information regarding the binding affinity, stoichiometry, competition and enthalpy of two or more interacting proteins.
Rotating cell‑based ligand binding assay using radioactivity or fluorescence, is a recent method that measures molecular interactions in living cells in real-time. This method allows the characterization of the binding mechanism, as well as K
d, k
on and k
off. This principle is being applied in several studies, mainly with protein ligands and living mammalian cells.
Single colour reflectometry
Single colour reflectometry (SCORE), formerly known as imaging Reflectometric Interferometry (iRIf) and 1-lambda Reflectometry, is a physical method based on interference of monochromatic light at thin films, which is used to investigate (bio-)mo ...
(SCORE) is a label-free technology for measuring all kinds of biomolecular interactions in real-time. Similar to BLI, it exploits interference effects at thin layers. However, it does not need a spectral resolution but rather uses monochromatic light. Thus, it is possible to analyse not only a single interaction but high-density arrays with up to 10,000 interactions per cm
2.
Genetic methods
The
yeast two-hybrid
Two-hybrid screening (originally known as yeast two-hybrid system or Y2H) is a molecular biology technique used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical interactions (such as bindi ...
and
bacterial two-hybrid screen investigate the interaction between artificial fusion proteins. They do not require isolation of proteins but rather use
transformation
Transformation may refer to:
Science and mathematics
In biology and medicine
* Metamorphosis, the biological process of changing physical form after birth or hatching
* Malignant transformation, the process of cells becoming cancerous
* Tran ...
to express proteins in
bacteria
Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were am ...
or
yeast
Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to consti ...
. The cells are designed in a way that an interaction activates the
transcription
Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including:
Genetics
* Transcription (biology), the copying of DNA into RNA, the fir ...
of a
reporter gene
In molecular biology, a reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. Such genes are called reporters because the char ...
or a reporter
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecule ...
.
Computational methods
Most PPI methods require some computational data analysis. The methods in this section are primarily computational although they typically require data generated by wet lab experiments.
Protein–protein docking
Macromolecular docking is the computational modelling of the quaternary structure of complexes formed by two or more interacting biological macromolecules. Protein–protein complexes are the most commonly attempted targets of such modelling, fol ...
, the prediction of protein–protein interactions based only on the three-dimensional protein structures from X-ray diffraction of protein crystals might not be satisfactory.
Network analysis Network analysis can refer to:
* Network theory, the analysis of relations through mathematical graphs
** Social network analysis, network theory applied to social relations
* Network analysis (electrical circuits)
A network, in the context of e ...
includes the analysis of interaction networks using methods of
graph theory
In mathematics, graph theory is the study of '' graphs'', which are mathematical structures used to model pairwise relations between objects. A graph in this context is made up of '' vertices'' (also called ''nodes'' or ''points'') which are conn ...
or statistical methods. The goal of these studies is to understand the nature of interactions in the context of a cell or
pathway
Pathway or pathways may refer to:
Entertainment
* ''The Pathway'' (novel), a 1914 work by Gertrude Page
*''The Pathway'', a 2001 album by Officium Triste
* ''Pathway'' (album), by the Flaming Stars
* ''Pathways'' (album) (2010), by the Dave Hol ...
, not just individual interactions.
References
{{DEFAULTSORT:Methods to investigate protein-protein interactions
Protein methods
Molecular biology techniques
Protein–protein interaction assays