Protein-Protein Interactions Support—Getting Started

It is the binding together of two or more proteins, often to carry out a biological function. As such, most biological activities are regulated directly or indirectly by protein-protein interactions.

The study of protein-protein interactions is vital to the understanding of how proteins function within the cell. Publication of the human genome and proteomics-based protein profiling studies catalyzed resurgence in protein interaction analysis. Characterizing the interactions of proteins in a given cellular proteome (often referred to as the “interactome”) will be the next milestone along the road to understanding the biochemistry of the cell. The ~30,000 genes of the human genome are speculated to give rise to 1 x 10⁶ proteins through a series of post-translational modifications and gene-splicing mechanisms. Although a population of these proteins can be expected to work in relative isolation, the majority are expected to operate in concert with other proteins in complexes and networks to orchestrate the myriad of processes that impact cellular structure and function. These processes include cell-cycle control, differentiation, protein folding, signaling, transcription, translation, post-translational modification and transport. Implications about function can be made via protein-protein interaction studies. These implications are based on the premise that the function of unknown proteins may be discovered if captured through their interaction with a protein target of known function.

Protein-protein interactions fundamentally can be characterized as stable or transient. Both stable and transient interactions can be either strong or weak. 

Stable interactions are those associated with proteins that are purified as multi-subunit complexes. The subunits of the complex can be identical or different. Hemoglobin and core RNA polymerase are two examples of stable multi-subunit complex interactions. Stable interactions are best studied by co-immunoprecipitation, pull-down or far-western methods.

Transient interactions are expected to control the majority of cellular processes. As the name implies, transient interactions are on/off or temporary in nature and typically require a specific set of conditions that promote the interaction. Transient interactions can be strong or weak, fast or slow. While in contact with their binding partners, transiently interacting proteins are expected to be involved in the whole range of cellular processes including protein modification, transport, folding, signaling, cell cycling, etc. Transient interactions can be captured by crosslinking or label transfer methods.

• DNA regulator complexes
• Antibody-antigen interactions (actin antibody with actin)
• Protein A/G with antibody

Here are some common methods for analyzing protein-protein interactions:

• Co-immunoprecipitation (Co-IP)
• Pulldown assays
• Yeast two-hybrid screening
• Crosslinking
• Label transfer
• Immunofluorescence/fluorescence resonance energy transfer (FRET)
• Far-western analysis
• Surface plasmon resonance: relates binding information to small changes in refractive indices of laser light reflected from gold surfaces to which a bait protein has been attached. Changes are proportional to the extent of binding. Special labels and sample purification are not necessary, and analysis occurs in real time.
• NMR (nuclear magnetic resonance): Method that can provide insights into the dynamic interaction of proteins in solution.
• Mass Spectrometry: Used in concert with affinity-based methods (such as co-IPs) to isolate binding partners and complexes and to identify the component proteins using standard mass spectral methods (e.g., MALDI-TOF and mass searching of bioinformatics databases). Heavy (deuterated) crosslinkers can be used in conjunction with mass spectroscopy to elucidate details about protein-protein interactions.
• X-ray crystallography: crystallization of the interacting complex allows definition of the interaction structure.

Visit our Protein Immunopreipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center for more information. 

Our qIP Luciferase assay kits have been discontinued but we do carry epitope-tagged (HA or c-Myc) and Tluc (TurboLuc luciferase)-tagged mammalian expression vectors, and qIP protein interaction assay reagents for these assay kits. 

We offer the Proquest™ Two-Hybrid System with Gateway™ Technology, which is a genetic method for detecting interactions between proteins in vivo in the yeast Saccharomyces cerevisiae. The ProQuest™ system can be used to:

• Test for interaction between two known proteins 
• Screen a library for novel proteins that specifically interact with a known bait

The ProQuest™ Two-Hybrid System offers a number of features to decrease false positives. They are listed below:

• Uses low-copy-number (ARS/CEN) vectors to control over-expression and increase reproducibility
• Contains three different reporter genes with independent promoters to rapidly weed out false positives
• Uses a reporter gene (URA3) that allows both positive and negative selection, which enables advanced two-hybrid techniques such as reverse two-hybrid
• An extended panel of yeast control vectors to aid in setting up the experiments and evaluate results
• Incorporation of the Gateway™ Technology to allow rapid and easy generation of bait and prey constructs, and to facilitate down-stream applications

The ProQuest™ Two-Hybrid System includes:

• Yeast expression vectors: pDEST™32 for generation of the bait plasmid containing the gene of interest in frame with the GAL4 DNA Binding Domain (GAL4 DBD), pDEST™22 for generation of the prey plasmid containing the second known gene of interest in frame with the GAL4 Activation Domain (GAL4 AD), and pEXP-AD502 to construct a cDNA or genomic library for identifying proteins (preys) that interact with the fusion protein (bait)
• Reagents for production of the expression clones containing GAL4 DBD and GAL4 AD fusion proteins
• A glycerol stock of yeast strain MaV203, which is the two-hybrid yeast strain used
• Positive and negative controls for the two-hybrid assay

The yeast strain provided in the ProQuest™ Two-Hybrid System is MaV203 that contains the following features:

• A set of non-reverting auxotrophic mutations: leu2 and trp1 to allow selection for the bait and prey fusion vectors, and his3 for growth upon induction of the reporter gene GAL1::HIS3
• Deletions of the GAL4 and GAL80 genes encoding GAL4 and its repressor GAL80, respectively. In the absence of GAL80, galactose is not required for activation of GAL4-inducible promoters
• Three stably integrated single-copy GAL4-inducible reporter genes: SPAL10::URA3 integrated at URA3; HIS3UAS GAL1::HIS3 integrated at LYS2; and GAL1::lacZ integrated at an unknown locus
• The recessive drug resistance marker cyh2R for plasmid shuffling

The genotype of MaV203 is as follows:

MaV203 (MATα, leu2-3,112, trp1-901, his3Δ200, ade2-101, gal4Δ, gal80Δ, SPAL10::URA3, GAL1::lacZ, HIS3UAS GAL1::HIS3@LYS2, can1R, cyh2R)

Note: The yeast strain MaV203 is unique to the ProQuest™ Two-Hybrid System. Other yeast strains used for two-hybrid analysis cannot be substituted.

This can definitely be done, especially if looking at multiple combinations of interactions. The two yeast strains used will have to be of different mating types (MATa and MAT alpha). Markers on the bait and prey plasmids will have to be used in selection of the diploids.

To avoid interference of endogenous GAL4 and GAL80 proteins, MaV203 must carry deletions of the GAL4 and GAL80 genes. As a result of deletion of these two genes, MaV203 cells grow more slowly compared to yeast strains containing the wild-type version of these genes. Note that the growth rate will depend on the type of growth medium used (e.g., YPD versus SC drop-out media).

No, the MaV203 cells are provided as a glycerol stock. Cells from this stock need to be streaked on a YPD plate for growth at 30 degrees C. Resulting colonies can be used to prepare competent cells for transformation as per the ProQuest™ Two-Hybrid System manual. Alternatively, ready-to-use MaV203 Competent Yeast Cells, Subcloning Scale (Cat. No. 11445-012) or Library Scale (Cat. No. 11281-011) are available for purchase from us.

(1) Suspend several colonies of MaV203 in 50 µL autoclaved, distilled water in a microcentrifuge tube and spread it onto the center of a 10-cm YPAD plate using an autoclaved loop or toothpick. Repeat procedure for a second YPAD plate. Incubate both plates for 18-24 h at 30 degrees C. 

(2) Scrape and completely suspend the cells (by brief vortexing and pipetting up and down) in 10 mL autoclaved, distilled water. Add a sufficient volume of this cell suspension to two 1-L flasks each containing 500 mL liquid YPAD medium to give an OD600 of ~0.1. Reserve approximately 10 mL YPAD medium to use as a blank in the spectrophotometer. 
Note: Perform serial 1:10 dilutions in water of the 10-mL cell suspension then determine the OD600 of each dilution to allow an estimate of cell suspension required to produce the desired OD of 0.1. Appropriate cell densities require that the measured OD be <1.0. Verify that the OD is ~0.1 after inoculation. Use plastic cuvettes for all OD600 measurements. 

(3) Shake (~250 rpm) at 30 degrees C until the OD600 reaches ~0.4 (usually ~5 h). Read the OD. 

(4) Prepare fresh: 
225 mL 1X TE/LiAc by combining 22.5 mL 10X TE, 22.5 10X LiAc, and 180 mL autoclaved H₂O. 
30 mL PEG/LiAc by combining 3 mL 10X TE, 3 mL 10X LiAc, and 24 mL 50% PEG-3350. 
200 µL carrier DNA by boiling sonicated herring sperm DNA or sonicated salmon sperm DNA (10 mg/mL) for 5 min and placing on ice until use. 

(5) Split each 500 mL of yeast cells into two conical 250-mL tubes and centrifuge at 3,000 x g for 5 min at room temperature. 

(6) Pour off the supernatants and gently suspend each pellet by pipetting up and down in 100 mL autoclaved, distilled water at room temperature. 

(7) Centrifuge at 3,000 x g for 5 min at room temperature. Pour the supernatant off of the centrifuged cells and suspend each cell pellet in 50 mL 1X TE/LiAc solution. 

(8) Centrifuge at 3,000 x g for 5 min at room temperature. Carefully pour off the supernatants and suspend each cell pellet in a final volume of 1 mL 1X TE/LiAc solution and pool all suspensions for a total of 4 mL. 

(9) Perform 30 transformations. Combine 4 mL of cells, 200 µL freshly boiled carrier DNA and 150 µg (~1 µg/µL) bait plasmid DNA and 150 µg (~1 µg/µL) plasmid-library plasmid DNA. Mix gently by pipetting up and down. Add 24 mL PEG/LiAc solution and mix gently, but completely. Aliquot into 30 autoclaved microcentrifuge tubes of 950 µL each.

(10) Incubate for 30 min in a 30 degrees C water bath. 

(11) Heat shock for 15 min in a 42 degrees C water bath. 

(12) Centrifuge in a microcentrifuge (6,000 - 8,000 x g) for 20-30 s at room temperature. Carefully remove the supernatant. 

(13) Gently suspend each pellet in 400 µL autoclaved, distilled water by pipetting up and down. 

(14) To estimate the total number of transformants, plate two dilutions of the transformation. Mix 10 µL of transformation with 90 µL autoclaved, distilled water. Plate 100 µL on a 10-cm SC-Leu-Trp plate (1:800 final dilution factor). Mix 10 µL of transformation with 990 µL autoclaved, distilled water. Plate 100 µL on a 10-cm SC-Leu Trp plate (1:8,000 final dilution factor).

The ProQuest™ Two-Hybrid System bait and prey expression vectors utilize the ADH1 promoter, which is generally considered to be a strong constitutive promoter. However, expression is repressed as much as 10-fold on non-fermentable carbon sources (Bartel PL (1996) Nat Genet 12:72-77).

3-AT is a toxic histidine precursor that is accumulated in cells lacking the HIS3 gene product. Since the his3 mutation is leaky, 3-AT is used to reduce the background growth of his3 cells. The amount of 3-AT used in plates typically ranges from 10 mM to 100 mM. Some investigators have reported using up to 400 mM 3-AT. To determine the appropriate concentration, you should perform a titration.

This can definitely be done especially if looking at multiple combinations of interactions. The 2 yeast strains used will have to be of different mating types (MATa, and MATalpha). Markers on the two plasmids will have to be used in selection of diploids. MaV103 can be used as mating partner for this purpose. MaV103 has the same genotype as MaV203 except they are MATa. MaV103 with bait vector will be LEU2+ and MaV203 with prey vector will be TRP1+. Therefore diploids can be selected on SC-leu-trp plates. For further details on this method, refer to Nature Genetics (1996), 12: 72-77; Vidal, M et al. (1996) PNAS 93:10315.

Visit our Protein Labeling, Crosslinking, and Modification Support Center for more information. 

Label transfer is a technology that can be used to discover new protein-protein interactions or to confirm putative interactions suggested by other methods, and to investigate the interface of the interacting proteins. In addition, the label transfer method is able to detect weak or transient protein interactions that often evade detection in co-immunoprecipitation and pulldown methods.

In a typical label transfer reaction, a purified bait protein is labeled with the chosen label transfer reagent. This labeled bait protein is allowed to interact with prey protein in vitro to form a complex. Once the complex has been formed, the reaction is exposed to UV light to activate a photo-reactive group and initiate the label transfer process by binding to the prey protein. Label transfer is completed by cleaving the spacer arm to release the bait protein, leaving the label attached to the interacting prey protein. A biotin label is especially useful in this process because it can be used both for purification and detection of the prey protein.

Currently, we offer sulfo-SBED Biotin Label Transfer Reagent (Cat. Nos. 33033 and 33034) as well as the Sulfo-SBED Biotin Label Transfer Kit for Western Blot Application (Cat. No. 33073).

The NHS-ester of sulfo-SBED biotin will react with primary amines on the bait protein. The photo reactive aryl azide primarily targets nucleophiles, such as –NH2, -SH and –OH, but in their absence, will react with any near neighbor molecule.

In the case of sulfo-SBED biotin there is a disulfide in the spacer arm of this tri-functional crosslinker that is cleaved with reducing agents such as DTT, BME or TCEP. As the disulfide lies between the contact point on the bait protein and the complex of the photoreactive group and biotin, reduction separates the bait protein from the prey protein; which is now biotin labeled. 

The far-western blotting technique is quite similar to western blotting as it is based on a protein-protein interaction between a prey protein or target and an interacting protein. In a western blot, an antibody is used to detect the corresponding antigen on a membrane; in a far-western blot, the detection is done using any non-antibody protein. In a classical far-western analysis, a labeled or antibody-detectable “bait” protein is used to probe and detect the target “prey” protein on the membrane. The sample (usually a lysate) containing the unknown prey protein is separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) or native PAGE and then transferred to a membrane. When attached to the surface of the membrane, the prey protein becomes accessible to probing. After transfer, the membrane is blocked and then probed with a known bait protein, which usually is applied in pure form. Following reaction of the bait protein with the prey protein, a detection system specific for the bait protein is used to identify the corresponding band on the membrane. 

Far-western blotting has been used to determine receptor-ligand interactions and to screen libraries for interacting proteins. With this method of analysis, it is possible to study the effect of post-translational modifications on protein-protein interactions, examine interaction sequences using synthetic peptides as probes and identify protein-protein interactions without using antigen-specific antibodies. See here for details.

Currently, we offer the Thermo Scientific Far-Western Blot Kit for Biotinylated-Proteins (Cat. No. 23500). This kit utilizes a biotinylated detection molecule to bind to the target on the membrane. The kit includes streptavidin-HRP, which binds to the biotin and allows for detection via chemiluminescence using one of our highly sensitive SuperSignal™ West Substrates. The kit also includes dilution buffer, PBS and pre-cut cellophane sheets, for those researchers who prefer to probe the membrane directly.

Note: Detection on the membrane is 5–10X more sensitive than in-gel detection.

We offer a wide variety of biotinylation reagents, which target primary amines, sulfhydryls (reduced thiols), nucleic acids and some that are non-specific in their target on a molecule. Our Biotinylation Reagent Selection Tool can help anyone from beginner to expert identify the best biotin labeling regent for their needs.