What is the difference between Yeast Two-Hybrid (Y2H) and Mammalian Two-Hybrid (M2H), and which one should I choose?

The primary difference lies in the host system used. Y2H uses yeast as a host, while M2H uses cultured mammalian cells. The choice depends on your specific research question. Our experts can help you decide the most appropriate system after discussing your specific protein of interest and research goals.

My protein is a membrane-bound receptor. Can I still use a two-hybrid system?

Absolutely. While traditional Y2H systems are not suitable for integral membrane proteins because the protein interaction must occur in the nucleus, we offer a specialized solution: the Split-Ubiquitin Y2H (DUALmembrane) system. This advanced platform is specifically designed to detect interactions between full-length membrane proteins directly within the cellular membrane. It has been successfully used to study a wide range of membrane proteins, including multi-pass transmembrane receptors, ion channels, and transporters, providing a powerful tool to explore this critical class of proteins.

What kind of libraries can I screen against my "bait" protein?

Creative Biolabs offers a wide range of high-quality, high-complexity prey libraries to maximize the chance of identifying physiologically relevant interactors. We provide: Standard cDNA Libraries: We have over 30 pre-made cDNA libraries from various species (human, mouse, rat, etc.) and tissues. Custom Library Construction: If a suitable library is not available, our team can construct a custom cDNA library from any specific cell line, tissue, or developmental stage relevant to your project. Normalized Libraries: To enhance the discovery of low-abundance transcripts, we can normalize the library to reduce the prevalence of highly expressed genes. Using a high-quality, complex library is critical for the success of a two-hybrid screen, and we ensure our libraries meet the highest standards.

I want to find a drug that breaks up a specific protein-protein interaction. Which service should I use?

For this application, our Reverse Yeast Two-Hybrid (rY2H) service is the ideal choice. This powerful system is specifically designed to screen for molecules or mutations that disrupt a known PPI. In the rY2H system, the interaction between your two proteins of interest activates a toxic reporter gene. Therefore, only yeast cells in which the interaction is broken by a compound from a chemical library can survive. This provides a direct and high-throughput method for identifying potential therapeutic inhibitors, making it an invaluable tool for the early stages of drug discovery.

What is the main difference between Y2H and rY2H?

Y2H is a discovery tool used to find unknown protein partners ("what does my protein bind to?"). rY2H is a screening tool used to find molecules that disrupt a known protein interaction ("what compound can break this specific protein pair apart?").

What types of protein interactions are suitable for the rY2H system?

The rY2H system is ideal for studying binary interactions that can be reconstituted in the yeast nucleus. It is compatible with proteins from various organisms and subcellular locations, though some modifications may be required for membrane or secreted proteins.

How do you handle false positives in the rY2H screen?

We employ a rigorous, multi-tiered validation strategy. This includes re-testing hits, performing counter-screens to identify compounds that are generally toxic to yeast or inhibit transcription/translation, and running dependency assays to ensure the effect is specific to the bait-prey interaction.

Can I screen my own compound library?

Absolutely. We are happy to work with our clients to screen their proprietary in-house libraries. Please contact us to discuss the logistics and formatting requirements.

High-Resolution Protein-Protein Interaction Analysis Service by X-Ray Crystallography

Platform Workflow Advantage Related Services FAQ Online Inquiry

Protein-protein interactions (PPIs) are the cornerstone of nearly every biological process, from signal transduction to immune responses. While numerous methods can detect PPIs, X-ray crystallography remains the undisputed gold standard for providing high-resolution, unambiguous structural data of protein complexes. Creative Biolabs harnesses the power of X-ray crystallography to provide an unparalleled view of the protein-protein interface, revealing the intricate network of contacts that govern molecular recognition, affinity, and specificity. Our end-to-end service empowers researchers to visualize these interactions with atomic precision, transforming abstract concepts into tangible, actionable data for structure-based drug design and fundamental research.

Discuss Your Project Today

Our State-of-the-Art PPI Crystallography Platform

At Creative Biolabs, we have built a world-class platform specifically optimized for the structural determination of protein-protein complexes, including those that are notoriously difficult to crystallize.

High-Throughput Crystallization Screening: We utilize robotic liquid handlers and a vast library of over 1,500 unique crystallization conditions (including commercial and proprietary screens) to maximize the chances of obtaining high-quality initial crystals.
Advanced Synchrotron Access: We leverage privileged access to cutting-edge synchrotron beamlines worldwide. These powerful X-ray sources are essential for collecting high-resolution diffraction data from even micro-crystals, ensuring the highest quality structural information.
Expertise with Challenging Complexes: Our seasoned scientists have a proven track record of success with challenging targets, including transient complexes, low-affinity interactions, and large multi-protein assemblies. We employ sophisticated protein engineering strategies (e.g., surface entropy reduction, fusion partners) to promote crystallization.
Fragment-Based Screening (FBS): For drug discovery projects, we can perform crystallographic fragment screening to identify novel small molecule binders that bind at the PPI interface, providing ideal starting points for lead development.
Integrated Computational Support: Our process is supported by advanced computational biology, from construct design and molecular dynamics simulations to sophisticated data processing and structural refinement, ensuring a comprehensive analysis.

Fig. 1 Major steps during X-ray crystallography. (OA Literature). Fig. 1 Flowchart representing the major steps during X-ray crystallography.¹

Our Comprehensive Service Workflow

We offer a fully managed, transparent workflow designed to deliver results efficiently. Each project is assigned a dedicated Ph.D.-level project manager to ensure clear communication and expert oversight at every stage.

Phase 1: Project Consultation and Feasibility Analysis

In-depth discussion with our structural biology experts.
Bioinformatic analysis of target proteins.
Development of a customized project strategy.

Phase 2: Protein Expression and Complex Formation

Codon optimization and gene synthesis.
High-level expression of individual proteins in a suitable system (E. coli, baculovirus, mammalian).
High-purity protein purification (>95%) using multi-step chromatography.
In vitro reconstitution and verification of the protein complex.

Phase 3: Crystallization and Data Collection

High-throughput screening for initial crystallization hits.
Meticulous optimization of crystal growth conditions.
Cryo-protection and crystal harvesting.
X-ray diffraction data collection at a synchrotron facility.

Phase 4: Structure Determination and Analysis

Diffraction data processing and scaling.
Phase determination (e.g., molecular replacement).
Iterative model building and refinement.
In-depth analysis of the PPI interface, key contacts, and structural features.

Phase 5: Deliverables and Reporting

High-purity purified protein complex.
Optimized crystallization conditions.
High-resolution, publication-quality final structure.
A comprehensive, detailed final report including all methods, data, analysis, and interpretation.

Why Choose X-Ray Crystallography for Your PPI Studies?

Choosing the right technology is critical for meaningful PPI analysis. X-ray crystallography offers distinct advantages that other methods cannot match.

Table 1. Comparison of X-ray Crystallography vs. other common PPI methods.

Feature	X-Ray Crystallography	Surface Plasmon Resonance (SPR)	Co-Immunoprecipitation (Co-IP)	Yeast Two-Hybrid (Y2H)
Primary Output	3D Atomic Structure	Binding Kinetics (ka, kd, KD)	Interaction Confirmation	Interaction Discovery
Resolution	Atomic (~1-3 Å)	N/A (Kinetics)	Low (Complex Level)	Low (Binary Interaction)
Interface Detail	Direct & Precise	Indirect Inference	No	No
Throughput	Low to Medium	Medium to High	High	Very High
Key Application	Structure-Based Drug Design, MOA	Affinity & Kinetics Screening	In Vivo Interaction Validation	Large-Scale Screening
False Positives	Very Low	Low	Medium	High

Explore Our Related Protein Interaction Services

Creative Biolabs offers a comprehensive suite of biophysical and structural biology services to complement your research. Explore our other platforms to build a complete picture of your molecular system. Explore our full range of PPI services here: Protein-Protein Interaction (PPI) Assay Services

Ready to illuminate your protein-protein interactions with atomic precision? Partner with the experts at Creative Biolabs. Our team is ready to discuss your project and design a strategy tailored to your specific research goals.

Contact Us Today for a Free Quote!

Frequently Asked Questions (FAQs)

Q: How much protein do I need to provide?

A: Typically, we require 5-10 mg of each purified protein at >95% purity to start a project. However, we also offer a full service starting from gene synthesis, so you don't need to provide any protein.

Q: What is the typical timeline for a project?

A: A standard co-crystallography project can range from 4 to 6 months, depending on the complexity of the target. We will provide a detailed timeline in our customized project proposal.

Q: What if my protein complex fails to crystallize?

A: Crystallization is a complex R&D process. While our success rate is very high, not all proteins will crystallize. In such cases, we offer multiple rescue strategies, including protein engineering and screening of orthologs. We can also pivot the project to another structural method like Cryo-EM, if suitable. Our milestone-based payment structure ensures you only pay for successfully completed stages.

Q: What resolution can I expect for the final structure?

A: Our goal is always to achieve the highest resolution possible. We typically deliver structures in the range of 1.5 - 3.0 Å, which is more than sufficient for detailed interface analysis and structure-based drug design.

Reference

Bijelic, Aleksandar, and Annette Rompel. "Polyoxometalates: more than a phasing tool in protein crystallography." ChemTexts 4.3 (2018): 10. Distributed under Open Access license CC BY 4.0, without modification. https://doi.org/10.1093/nar/gkx1173

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