The biological department of Shanghai Medicilon has extensive experience in the field of in vitro biology, through enzyme level measurement, cell level measurement, cell biology, biochemistry, in vitro isotope measurement, stable cell line establishment, gene knockout, RNAi and MicroRNA Technology, etc., to provide a complete set of biological services.
Medicilon Enzyme Level Determination Service
Enzyme level determination
Multiple technology platforms
AlphaScreen analysis test
Z’-LYTE analysis test
Adapta® Universal Kinase Analysis
Kinase-Glo® Kinase Luminescence Detection and Analysis
ADP-GloTM kinase assay
HTRF homogeneous time-resolved fluorescence detection analysis
Diversity targets and applications
Enzyme activity, also known as enzyme activity, refers to the ability of enzymes to catalyze chemical reactions. The enzyme activity can be expressed by the speed of each reaction under certain conditions. The faster the reaction rate, the higher the enzyme activity in a given enzyme solution or tissue extract.
Enzyme activity detection
Method for measuring enzyme activity
According to different choices of enzymatic reaction time, it is divided into: fixed world method, continuous monitoring method
①Fixed world method: Determine the amount of substrate decrease or product increase within a period of time after the enzymatic reaction starts. Advantages: Simple Disadvantages: It is difficult to determine whether the enzymatic reaction in the reaction time period is in the linear phase.
②Continuous detection method: to determine the amount of change of substrate or product with time, also known as rate method. Advantages: can dynamically observe the progress of the enzymatic reaction, can clearly find the linear phase of the reaction, the results are accurate and reliable, the amount of specimens and reagents is small, and the determination can be completed in a short time. Disadvantages: high environmental requirements, precise control of reaction conditions such as temperature, PH value and substrate concentration, and the instrument has a constant temperature device and automatic detection function.
Principles of enzyme activity determination
Direct measurement of reaction product, enzymatic reaction rate and its measurement-expressed in terms of changes in the concentration of reactant or product per unit time.
Enzyme activity determination method
Common enzyme activity detection methods include: coupling reaction, chemical determination method, radioisotope method, pressure measurement method, electrode method, HPLC method, etc.
Factors affecting enzyme activity test
Generally, the substrate is excessive, so that the enzyme can fully bind to the substrate and exert its catalytic function.
②Type and concentration of substrate and cofactor
- Choose the appropriate substrate type and concentration:
If the measured enzyme is not specific, it can act on a variety of substrates. First, you need to decide which type of substrate to choose as the substrate for testing this enzyme. After determining the type of substrate, the important issue is to choose the appropriate concentration of substrate.
- Types and concentrations of cofactors and activators:
In a broad sense, any substance that can promote the enzyme and reactant into an activated state to accelerate the enzyme-catalyzed reaction can be called a cofactor, which includes a wide range of substances.
③ The optimal pH of the reaction system, the type and concentration of the buffer
Under the condition that the other conditions of the enzyme reaction are unchanged, the relationship between various types of enzyme activity and pH can be obtained at different pHs, and the pH at the highest enzyme activity is called the optimal pH.
The buffer contains a lot of ions, or affects the configuration of the enzyme protein, or affects the degree of dissociation of the substrate, etc., thereby affecting the enzyme activity.
When measuring enzymes, enzymes and substrates are required to work at a certain temperature for a period of time. During this period, the temperature may inactivate the enzymes. Different enzymes vary greatly in this regard. At present, more and more routine laboratories use 37 ℃, the temperature rises, the reaction speed is fast, and the sensitivity is high.
No matter what kind of temperature measurement enzyme is used, since the enzyme reaction is greatly affected by temperature, the temperature change of the reaction system should be controlled within ±0.1℃ within the measurement time. It is not appropriate to use a domestic ordinary constant temperature water bath to measure enzyme activity, and an advanced constant temperature water bath with a stirrer should be used.
⑤Other factors affecting enzyme activity
Among other factors, the most important thing for the determination of enzyme activity is the action of inhibitors.
Experimental design and precautions for enzyme activity detection
Parallel test: In the determination of enzyme activity, in order to eliminate system and operation errors, normal and abnormal controls must be set for each determination, the sample to be tested is set in duplicate, the average value of the results is taken, and the normal value and control tube of the laboratory are integrated the result of. Frequently, enzymes that are easily inactivated are used as reference enzymes for simultaneous determination. Only when the activity of the reference enzymes is normal can the results be reliable and exclude false positives.
Prevent enzyme inactivation: This is the key to the stability and success of the experiment. Cell separation, ultrasonication, centrifugation and other operations should be performed at 4°C, and specimens should be processed immediately after collection.
Enzyme activity determination reaction termination method
① Change the pH value to make the protein denature and precipitate
②Hot 100 degrees
③Stop the reaction with 1% SDS, note that protease K etc. are still active under SDS.
④If metal ions participate in the reaction, EDTA can be added
⑥ A large amount of non-radioactive matrix can be added during the radiometric determination
The above is about enzyme activity measurement, enzyme activity detection, etc. The information comes from the official website.