Enzymatic assay
- Lance Assay
- Alphascreen Assay
- Z’-LYTE Assay
- Adapta Assay
- Kinase-Glo Assay
- ADP-Glo Assay
- Ligand Binding Assay
- ELISA Assay
- HTRF Assay
Enzyme activity assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition.
Enzyme units: Amounts of enzymes can either be expressed as molar amounts, as with any other chemical, or measured in terms of activity, in enzyme units.
Medicilon provides various enzyme activity assays for kinases, phosphatases, proteinases, deacetylase, peptidase, esterase, and other enzymes. Our line of well-characterized immunoassays and biochemical kits ensures accurate and reproducible results.
Enzyme is a large category of bio-molecules that catalyze various biological processes including metabolic processes, cellular signaling and regulation, cell division and apoptosis. Enzymatic reactions convert substrate molecules into chemically modified molecules products with high specificity and accuracy. These modifications include proteolytic digestion, glycolytic degradation, phosphorylation, acylation, glycosylation or oxidative and reductive processes.
Our decades of experience in the design and manufacture of active enzymes and their substrates supports development of an ever-expanding portfolio of biochemical assays. We provide services on enzymatic assays to assist scientists focused on kinase research:
- kinetic evaluation of enzyme activity and inhibition
- inhibitor screening and validation
- IC50 determination of drug compounds
- enzyme-inhibitor studies analyzing mode of action
- investigations on reversibility of enzyme inhibition
- substrate profiling analyzing selectivity of enzymes
Our enzyme activity assays involve the immunocapture of the protein of interest first followed by measurement of substrate or product turnover. These employ only monoclonal antibodies that have been screened to isolate the enzyme in active form and in complexes that likely represent their associations in situ. There are many advantages. The enzyme is separated from other proteins that use or produce the same substrates or products screened and this allows for simple assays such as utilization of ATP, NADH, pyruvate and etc., which are subject to multiple reactions in vivo. For most of the enzymes for which we have activity assay kits, we also provide Sandwich ELISA assays of quantity. By combining the two, it is possible to generate specific activity data. Furthermore, because the enzyme is separated from other cellular components, the post-translational modifications (PTMs) it contains at the time of assay can be measured e.g. by subsequent mass spectrometry or reaction to PTM specific antibodies.
Method of Enzyme Assay
- Spectrophotometric Methods
- Fluorescence Methods
- Sampling Methods
- Manometric Methods
- Eletrode Methods
- Polarimetric Method
Types of Assay
All Enzyme Assays measure either the consumption of substrate or production of product over time. A large number of different methods of measuring the concentrations of substrates and products exist and many enzymes can be assayed in several different ways. Biochemists usually study enzyme-catalysed reactions using four types of experiments:
(1) Initial Rate Experiments
When an enzyme is mixed with a large excess of the substrate, the enzyme-substrate intermediate builds up in a fast initial transient. Then the reaction achieves a steady-state kinetics in which enzyme substrate intermediates remains approximately constant over time and the reaction rate changes relatively slowly. Rates are measured for a short period after the attainment of the quasi-steady state, typically by monitoring the accumulation of product with time. Because the measurements are carried out for a very short period and because of the large excess of substrate, the approximation free substrate is approximately equal to the initial substrate can be made. The initial rate experiment is the simplest to perform and analyze, being relatively free from complications such as back-reaction and enzyme degradation. It is therefore by far the most commonly used type of experiment in enzyme kinetics.
(2) Progress Curve Experiments
In these experiments, the kinetic parameters are determined from expressions for the species concentrations as a function of time. The concentration of the substrate or product is recorded in time after the initial fast transient and for a sufficiently long period to allow the reaction to approach equilibrium. We note in passing that, while they are less common now, progress curve experiments were widely used in the early period of enzyme kinetics.
(3) Transient Kinetics Experiments
In these experiments, reaction behavior is tracked during the initial fast transient as the intermediate reaches the steady-state kinetics period. These experiments are more difficult to perform than either of the above two classes because they require rapid mixing and observation techniques.
(4) Relaxation Experiments
In these experiments, an equilibrium mixture of enzyme, substrate and product is perturbed, for instance by a temperature, pressure or pH jump, and the return to equilibrium is monitored. The analysis of these experiments requires consideration of the fully reversible reaction. Moreover, relaxation experiments are relatively insensitive to mechanistic details and are thus not typically used for mechanism identification, although they can be under appropriate conditions.
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Medicilon Enzyme Activity Testing Service
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