Methods to Detect Protein-Protein Interaction
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The document discusses methods for detecting protein-protein interactions (PPIs), emphasizing their importance in understanding biological functions and cellular organization. Common techniques include co-immunoprecipitation, pull-down assays, and label transfer assays, each with its own mechanisms for identifying interacting proteins. Additionally, it highlights methods for proteome-scale mapping of interactions, such as yeast two-hybrid and affinity purification with mass spectrometry.
Methods to Detect Protein-Protein Interaction
- 1. Methods for Detection of Protein-Protein Interactions
- 2. Protein–Protein Interactions • Proteins affect a number of biological functions，which are often depend on macromolecular interactions. It has been estimated that more than 80% of proteins do not operate alone but in complexes. • The analysis of PPIs is able to help us understand cellular organization, process, and functions. • PPIs analysis may discovery unique and unforeseen functional roles for well-known proteins. And it can discover previously unknown proteins by association known proteins.
- 3. Approaches to investigate PPIs Types of Approaches Protein-Protein Interactions
- 4. Common methods to analyze protein–protein interactions Co- immunopr ecipitation (Co-IP) Pull-down Assay Label Transfer Assay
- 5. Common methods to analyze protein–protein interactions Co-immunoprecipitation (Co-IP) Protein-Protein Interactions Cell lysis Incubation with antibody Removal of unbounded proteins Western blot/mass spectrometry anslysis Targeting a known protein with a specific antibody can pull the entire protein complex, therefore, the unknown members of the complex can be identified. It is suitable for proteins involved in the complex bind to each other tightly. It reveals direct and indirect interactions, so results may indicate that two proteins interact directly or may interact via one or more bridging molecules.
- 6. Common methods to analyze protein–protein interactions Pull-down Assay Protein-Protein Interactions • A bait protein is tagged and captured on an immobilized affinity ligand specific for the tag. • When the cell lysate is incubated with an immobilized bait protein, proteins binding to the bait protein can be captured and “pulled down”. • There are many types of tags for the bait of pull-down assay, such as GST, poly-His, and biotin, etc. The affinity ligand used to immobilize bait include glutathione, nickel and chelate complexes, and streptavidin. GST-tag Bait Protein Prey Protein Prey Protein
- 7. Common methods to analyze protein–protein interactions Label Transfer Assay Protein-Protein Interactions • The protein of interest is tagged with a label transfer reagent (LTR, composed of a label radical and and a photoreactive group). • After the crosslinking reaction, the label is transferred to an interacting partner and allowing its determination by multiple methods, like western blot analysis, protein sequence analysis, and mass spectrometry. Label radical group Photoreactive group Reactive group Protein1 Protein2 Introduce interacting protein Active photoreactive group with light Transfer label by reducing agent
- 8. Methods for proteome-scale interactome maps Mapping protein-protein interactions on a proteomics scale reveal the macromolecular connections that underlie the biology of the cell. • Binary mapping by yeast two-hybrid (Y2H) • Affinity purification and mass spectrometry (AP-MS) • Co-fractionation and mass spectrometry Cafarelli T M, et al. Mapping, modeling, and characterization of protein–protein interactions on a proteomic scale. Current opinion in structural biology, 2017, 44: 201-210. Protein-Protein Interactions
- 9. Binary mapping by yeast two-hybrid (Y2H) Cafarelli T M, et al. Mapping, modeling, and characterization of protein–protein interactions on a proteomic scale. Current opinion in structural biology, 2017, 44: 201-210. Methods for proteome-scale interactome maps It detects direct physical interactions between two proteins by the reconstitution of a transcription factor that activates reporter gene expression and promotes yeast cell survival on appropriate selective media. Protein-Protein Interactions
- 10. Co-complex maps Cafarelli T M, et al. Mapping, modeling, and characterization of protein–protein interactions on a proteomic scale. Current opinion in structural biology, 2017, 44: 201-210. Methods for proteome-scale interactome maps AP-MS: In this method, epitope tags are fused to bait proteins, and proteins associated with the bait proteins are purified and identified by mass spectrometry. Co-fractionation followed by MS: chromatographic and other biochemical separation methods of cell extracts are carried out to generate hundreds to thousands of fractions that are analyzed by mass spectrometry. Protein-Protein Interactions
- 11. Our Services Protein-Protein Interactions Services 01Co-immunoprecipitation 05Far-Western Blot Analysis 02 Pull-Down Assay 03 Crosslinking Protein Interaction Analysis 04 Label Transfer Protein Interaction Analysis At Creative Proteomics, our team of experts with extensive experience can help you understand what you are trying to investigate and give you the most appropriate solutions about protein and protein interactions. Protein-Protein Interactions
- 12. THANK YOU Please contact us for more information Web: Email: www.creative-proteomics.com info@creative-proteomics.com
Editor's Notes
- #2: Hello, welcome to watch the creative proteomics . Today, we are going to talk about methods for Detection of protein-protein interactions.
- #5: Since the methods to investate PPIs are too numerous to describe here, we focus on some common methods in the following parts, including Co-immunoprecipitation, Pull-down Assay, and Label Transfer Assay.
- #12: At Creative Proteomics, our team of experts with extensive experience can help you understand what you are trying to investigate and give you the most appropriate solutions about protein and protein interaction. We can provide protein-protein interaction services based on different methods, including: Co-immunoprecipitation (co-IP) Pull-Down Assay Crosslinking Protein Interaction Analysis Label Transfer Protein Interaction Analysis Far-Western Blot Analysis