Enzyme Activity Assay Services

Q: How do I choose between in vitro and cell-based kinase assays?

In vitro kinase assays use purified enzymes and provide precise biochemical potency (IC50) with rapid turnaround, making them ideal for primary screening and SAR studies. Cell-based kinase assays evaluate compound activity within living cells, capturing membrane permeability, intracellular ATP competition, and metabolic stability. Cell-based data are generally more predictive of in vivo efficacy. We recommend using both platforms sequentially—biochemical screening followed by cellular validation—to maximize confidence in lead compounds.

Cell-Based Kinase Assays

General Enzyme Activity Assays

In Vitro Protein Kinase Assays

CYP Inhibition Assay Using LC-MS/MS

Single-Cell Enzyme Assays

Enzyme Activator & Inhibitor Screening

Enzyme activity assay

A fundamental task of proteins is to act as enzymes—catalysts that increase the rate of virtually all the chemical reactions within cells. Enzymes play important roles in numerous physiological activities, including metabolic processes, cellular signaling and regulation, cell division and apoptosis. In addition, many of them become critical targets for diseases treatments. For example, matrix metalloproteinase (MMP) plays critical role in anti-tumor metastasis. Understanding how well a key enzyme functions in a physiological process often reveals early signs of disease that has a long latency period. Thus, enzyme activity assay is crucial that it can provide data for reflecting activity and validating activator and inhibitor against disease-related enzyme, which is great significance in disease diagnosis, novel drug development and clinical therapy.

As one of the well-known service providers in biotechnology field, Profacgen provides comprehensive Enzyme Activity Assay Services that utilize advanced technology and proven expertise to help customers meet their analysis needs. Our enzyme activity assay team has rich experience in the field, and can provide personalized activity analysis and activator and inhibitor screening services against all kinds of enzymes.

Overview: Enzyme Activity Evaluation

Profacgen's enzyme activity assay platform is designed to deliver quantitative, reproducible data across the full spectrum of enzyme classes and applications. Our integrated approach combines multiple detection modalities with customized assay development to support projects ranging from single-enzyme kinetic characterization to high-throughput screening of compound libraries.

Our core capabilities encompass three interconnected domains:

Enzyme activity evaluation: Quantitative measurement of catalytic turnover, substrate affinity, and reaction kinetics under physiologically relevant or optimized conditions
Inhibitor and activator screening: Systematic evaluation of small-molecule modulators to identify hits, determine potency (IC₅₀/EC₅₀), and elucidate mechanism of action
Kinetic characterization: Determination of K_m, V_max, k_cat, and K_i to support structure–activity relationship studies and regulatory submissions

Our Enzyme Assay Capabilities

Profacgen offers a broad portfolio of specialized enzyme assay services, each tailored to specific enzyme families, detection requirements, and project goals.

Kinase Assays

Protein kinases represent one of the most extensively pursued target classes in drug discovery. We provide both biochemical and cell-based kinase assay platforms:

In Vitro Protein Kinase Assays: Radiometric, fluorescence, luminescence, mobility shift, and ELISA-based formats for purified enzyme inhibition and ATP competition studies.
Cell-Based Kinase Assays: Pathway activation, intracellular phosphorylation, and cellular potency evaluation using phospho-specific antibodies, reporter systems, and flow cytometry.

CYP Inhibition

Cytochrome P450 enzymes mediate the metabolism of over 90% of marketed drugs. Our LC-MS/MS-based CYP inhibition platform supports:

CYP Inhibition Screen Assay Using LC-MS/MS: Reversible and time-dependent inhibition assessment against CYP3A4, CYP2D6, CYP2C9, CYP1A2 and CYP2C19 using the regulatory agencies' recommended probe substrates.

Single-Cell Enzyme Analysis

For projects requiring resolution of cellular heterogeneity, we offer:

Single-Cell Enzyme Assays: Microfluidic droplet encapsulation, fluorescence imaging, and flow cytometry-based detection for rare subpopulation identification and immune profiling.

Substrate Conversion Assays

Our general enzyme activity platform supports diverse enzyme classes through tailored substrate and detection strategies:

General Enzyme Activity Assays: Customized assays for proteases, phosphatases, oxidases, hydrolases, and other enzyme families with versatile colorimetric, fluorometric, and luminescent readouts.

Activator and Inhibitor Screening

For drug discovery programs seeking novel modulators, we provide:

Enzyme Activator & Inhibitor Screening: High-throughput screening, focused library evaluation, and lead optimization with hit identification, SAR analysis, and mechanistic classification.

Detection Methods

Profacgen employs multiple detection platforms to match the catalytic properties of each enzyme and the sensitivity requirements of the project:

Method	Principle	Best Suited For	Key Advantages
Fluorescence	Fluorogenic substrate cleavage or FRET-based signal generation; intensity proportional to product formation	Proteases, phosphatases, hydrolases; real-time kinetics; HTS	High sensitivity, homogeneous format, multiplexing capability
Luminescence	Coupled luciferase reactions convert enzymatic products into light; signal inversely proportional to ATP consumption for kinases	Kinases, ATPases; ultra-low background applications; 384/1536-well HTS	Extremely wide dynamic range, excellent signal-to-noise, miniaturization-friendly
LC-MS/MS	Direct quantification of substrate and product masses enables label-free detection with high specificity	CYP inhibition, complex substrates, non-peptide molecules, metabolite identification	Label-free, definitive structural confirmation, multiplexed detection, regulatory acceptance
Colorimetric	Chromogenic substrates produce colored products absorbable at defined wavelengths	Routine kinetic assays, cost-sensitive projects, educational settings	Low cost, no specialized equipment, straightforward interpretation
Radioactive Assays	[γ-³²P]- or [γ-³³P]-labeled ATP enables direct phosphate incorporation measurement by scintillation counting	Kinases, methyltransferases, acetyltransferases; gold-standard validation	Highest sensitivity and accuracy, direct mechanistic readout

Applications

Our enzyme activity assay services support a wide range of research and development objectives across biotechnology and pharmaceutical industries:

Drug screening: Primary and secondary screening of small-molecule libraries, natural products, and fragment collections to identify enzyme inhibitors and activators with therapeutic potential.
Enzyme engineering: Directed evolution and rational design campaigns leveraging kinetic data to optimize catalytic efficiency, substrate specificity, and thermostability.
Metabolic studies: CYP inhibition and induction profiling to predict drug–drug interactions, guide dosing strategies, and support regulatory submissions.
Biomarker validation: Quantitative enzyme activity measurements in clinical samples for disease diagnosis, patient stratification, and treatment monitoring.

Inquiry

Why Choose Profacgen?

Comprehensive Enzyme Coverage: From kinases and phosphatases to proteases, oxidases, and hydrolases—our platform supports all major enzyme families with specialized assay formats
Multiple Detection Platforms: Fluorescence, luminescence, LC-MS/MS, colorimetric, and radiometric assays matched to sensitivity, throughput, and regulatory requirements
Custom Assay Development: Tailored protocols for novel or poorly characterized enzymes, from substrate identification through full validation and SOP delivery
High-Throughput Compatibility: Automated liquid handling and 384/1536-well formats for efficient screening of large compound libraries
Experienced Scientific Team: Deep expertise in enzymology, assay development, and drug discovery workflows, with proven track records across kinase, CYP, and general enzyme programs
Integrated Data Analysis: Automated curve fitting, statistical analysis, and structured reporting to accelerate decision-making and regulatory readiness

Frequently Asked Questions (FAQs)

Q: What types of enzyme assays does Profacgen offer?

A: Profacgen provides a comprehensive enzyme assay platform covering kinase assays (in vitro and cell-based), CYP inhibition screening using LC-MS/MS, single-cell enzyme analysis, general substrate conversion assays for proteases/phosphatases/oxidases/hydrolases, and high-throughput activator/inhibitor screening. We support multiple detection formats including fluorescence, luminescence, colorimetric, radiometric, and mass spectrometry-based methods.

Q: How do I choose between in vitro and cell-based kinase assays?

A: In vitro kinase assays use purified enzymes and provide precise biochemical potency (IC₅₀) with rapid turnaround, making them ideal for primary screening and SAR studies. Cell-based kinase assays evaluate compound activity within living cells, capturing membrane permeability, intracellular ATP competition, and metabolic stability. Cell-based data are generally more predictive of in vivo efficacy. We recommend using both platforms sequentially—biochemical screening followed by cellular validation—to maximize confidence in lead compounds.

Q: What is the difference between reversible and time-dependent CYP inhibition?

A: Reversible CYP inhibition is an equilibrium process where the inhibitor binds to the enzyme active site without permanent modification; activity is restored upon dilution. Time-dependent inhibition (TDI) involves CYP-mediated bioactivation to a reactive metabolite that covalently modifies the enzyme, resulting in irreversible or quasi-irreversible inactivation. TDI carries greater clinical drug–drug interaction risk because the inhibitory effect persists after the parent drug is cleared. Our LC-MS/MS platform evaluates both mechanisms using pre-incubation studies and k_inact/K_I parameter determination.

Q: Can you develop custom assays for enzymes not in your standard catalog?

A: Absolutely. Custom assay development is one of our core strengths. We work with clients to identify suitable substrates, optimize reaction conditions (pH, temperature, cofactors), select the most appropriate detection platform, and validate the assay for precision and robustness. A qualified standard operating procedure (SOP) and protocol transfer support are delivered upon completion. Typical custom development timelines range from 4–6 weeks depending on substrate availability and complexity.

Q: What detection method is best for my enzyme assay project?

A: The optimal detection method depends on your enzyme's catalytic properties, substrate characteristics, sensitivity requirements, and throughput needs. Fluorescence and luminescence assays offer high sensitivity and are ideal for high-throughput screening. LC-MS/MS provides definitive structural confirmation and is preferred for complex substrates, metabolite identification, and regulatory studies. Colorimetric methods are cost-effective for routine kinetic work. Radiometric assays remain the gold standard for sensitivity in kinase and transferase studies. Our scientific team will recommend the most suitable platform during project consultation.

Q: How long does a typical enzyme assay project take?

A: Standard assays using validated protocols can be completed within 2–3 weeks. Custom assay development typically requires 4–6 weeks. High-throughput screening campaigns are scheduled based on library size and replicate requirements, generally 4–8 weeks. CYP inhibition screens (single-point, five isoforms) take 2–3 weeks, while full IC₅₀ determinations with time-dependent inhibition assessment require 4–6 weeks. We provide detailed timelines during consultation and offer expedited processing for time-critical programs.

Profacgen's enzyme activity assay team, based on many years of experience in basic and applied research in drug development, disease diagnosis and enzyme engineering, is confident in delivering the reliable assay results to help you succeed in your research projects. Thus, please do not hesitate to contact us for more details of our enzyme activity assay services. Our expert representatives are available 24 hours a day, Monday through Friday, to assist you.