Substrates for Peroxidase
Substrates for Alkaline Phosphatase
Substrate for Luciferase
Substrates for β-Galactosidase
Substrates for Peroxidase
Peroxidase is an enzyme that oxidizes various compounds in the presence of hydrogen peroxide. The enzyme is used for quantifying and the qualifying of many compounds including biogenic substances. Moreover, peroxidase is applied, due to its stability, to perodixase-labeled antibodys etc. and they are used in the fields of biochemistry, immunology, molecular biology.
Colorimetry is used for quantifying and qualifying peroxidase activity. A variety of chromogenic substrates are used for the purpose. The generally-used colorimetric substrates and their usage are summarized in the table as follows.
Generating soluble dye
(For ELISA etc.)
P1805
|
T1764
|
D3865
|
T2573
|
A2254
|
A0257
|
A2291
|
A2166
|
D3868
|
D3866
|
Generating insoluble dye
(For immunohistochemistry, Southern-blotting, Western-blotting etc.)
N0864
|
D3756
|
D3757
|
A2167
|
C2291
|
D3931
|
D3864
|
D3893
|
Substrates for Alkaline Phosphatase
Alkaline phosphatase is an enzyme that hydrolyzes phosphorylated substances under alkaline conditions. As the enzyme activity in serum originates in the liver, small intestines, and the osseous tissue, the activity is made to one of the disease markers of these tissues in the field of diagnosis. On the other hand, alkaline phosphatase is widely applied as a research reagent in the field of biochemistry and molecular biology and used to detect the antigen-antibody reaction as a labeling enzyme for antibodies. BCIP and NBT are substrates that are frequently employed for detecting the labeled enzyme on the blotting-experiment etc. BCIP’s phosphoester bond is hydrolyzed by the enzyme and converted to a blue compound. NBT is reduced, by the linkage of the reaction described above, and converted to a blue purple compound which forms an insoluble precipitate. The precipitate gives a clear signal for an assay.
I0781
|
B1239
|
C2250
|
D0844
|
Substrate for Luciferase
Luciferase is an enzyme that catalyzes luminescent reactions in bioluminescent organisms. Luciferase from the beetle catalyzes the two steps of luciferin oxidation in the presence of ATP, Mg2+, and oxygen molecules.
The beetle's bioluminescence system, for example, the firefly system, is applied to the analysis of biological materials at the forefront of life sciences. For instance, the transcription activity of a special gene in cells is frequently assayed as an index for analysis and evaluation of the toxicity or medicinal effect of chemicals.
As the method of measuring gene transcription, i.e., gene expression assay (reporter assay) system, luciferase is an important tool in today's life sciences.
A5030
|
| A5030 | D-(-)-Luciferin [Chemiluminescence Reagent] |
Substrates for β-Galactosidase
β-Galactosidase is an enzyme that hydrolyzes lactose to glucose and also acts broadly on allyl and alkyl β-D-galactosides. 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal), which is a substrate of β-galactosidase, is hydrolyzed to galactose and 5-bromo-4-chloro-3-hydroxyindole by the action of the enzyme. 5-bromo-4-chloro-3-hydroxyindole generated by the reaction is oxidized and converts to 5,5’-bromo-4,4’-dichloroindigo, which forms a blue insoluble precipitate. The chromogenic signal of the precipitate offers the detection of the enzymatic activity with high sensitivity. Thus, X-Gal is widely used for assays, for example, color selection (Blue-white selection) of genetically-modified organisms with an introduced lacZ gene, in molecular biology, biochemistry, and histochemistry.
N0418
|
N0616
|
B3470
|
B3201
|
B3469
|
C2371
|
■Typical Procedure Blue-white selection of E. coli expressing lacZ gene
・ 100mM IPTG solution: IPTG (0.238g) is dissolved in 1mL of sterile water and the solution is sterilized with filtration and stored at -20°C before use.
・ 20mg/mL X-Gal solution: X-Gal (40mg) is dissolved into 1mL of N,N-dimethylformamide. The solution is stored under dark at −20°C before use.
・ 100mM IPTG solution (40μL) and 20mg/mL X-Gal solution (40μL) is dropped onto LB-agar medium (10cm) and is spread on the medium with glass beads or with a spreader.
・ Appropriate amount of the gene-introduced E. coli cells is inoculated on the agar-medium with glass beads or with a spreader.
・ The cells are cultivated at 37°C over night, and the colonies grown on the agar-medium are counted.*
*When lacZ-expression plasmid vectors for gene cloning are used, some genes would be inserted into the vectors from white colonies.
Literature
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