For more information, you can visit https://www.creative-proteomics.com/services/protein-post-translational-modification-analysis.htm. In this video, we introduce some commonly used methods to detect PPIs and techniques for proteome-scale interactome maps.
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.
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
Notas del editor
Hello, welcome to watch the creative proteomics . Today, we are going to talk about methods for Detection of protein-protein interactions.
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.
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