9001-78-9 Usage
Description
EC 3.1.3.1, also known as Alkaline Phosphatase, is an enzyme that catalyzes the hydrolysis of 5'-terminal monophosphate groups from DNA and RNA. It plays a crucial role in preventing self-annealing of nucleic acid fragments and is widely used in various molecular biology applications.
Used in Molecular Biology:
EC 3.1.3.1 is used as a dephosphorylating enzyme for preventing recircularization of cloning vector DNA during ligation, dephosphorylating DNA prior to end-labeling using T4 Polynucleotide Kinase, and treating dNTPs in PCR reactions prior to sequencing or SNP analysis.
Used in Enzyme-Linked Immunosorbent Assay (EIA):
Alkaline Phosphatase, calf intestine, EIA Grade is used as a detection enzyme in EIA for dephosphorylation of nucleic acids, providing a sensitive and specific signal for the detection of target molecules.
Used in Probe Design:
EC 3.1.3.1 is used for 5'-nucleic acid targeting in the design of probes, enabling specific and efficient hybridization to the target sequence.
Biochem/physiol Actions
Alkaline phosphatase is a model enzyme for understanding phosphomonoesterase. It is used in various biochemical methods and enzyme linked immunosorbent assay (ELISA).
Purification Methods
The E.coli supernatant in sucrose (20%, 33mM) in Tris-HCl pH 8.0 is purified through a DEAE-cellulose column and recrystallised. To the column eluates in 0.125M NaCl is added MgCl2 (to 0.01M) and brought to 50% saturation in (NH4)2SO4 by adding the solid (0.20g/mL). The mixture is centrifuged to remove bubbles and is adjusted to pH 8.0 (with 2N NaOH). Saturated (NH4)2SO4 at pH 8.0 is added dropwise until the solution becomes faintly turbid (~61% saturation). It is set aside at ~25o for 1hour (turbidity will increase). The mixture is placed in an ice bath for several minutes when turbidity disappears and a clear solution is obtained. It is then placed in a large ice bath at 0o (~5L) and allowed to warm slowly to room temperature in a dark room whereby crystals are formed appearing as a silky sheen. The crystals are collected by centrifugation at 25o if necessary. The crystalline solutions are stable at ~25o for many months. They can be stored at 0o, but are unstable when frozen. Cysteine at 10-3M and thioglycolic acid at 10-4M are inhibitory. This is reversed on addition of Zn2+ ions. Many organic phosphates are good substrates for this phosphatase. [Molamy & Horecker Methods Enzymol 9 639 1966, Torriani et al. Methods Enzymol 12b 212 1968, Engstrom Biochim Biophys Acta 92 71 1964.] Alkaline phosphatase from rat osteosarcoma has been purified by Me2CO precipitation and chromatography on DEAE-cellulose, Sephacryl S-200, and hydroxylapatite. [Nair et al. Arch Biochem Biophys 254 18 1987.] Phosphoproteins (various). These are purified by adsorbing onto an iminodiacetic acid substituted agarose column to which are bound ferric ions. This chelate complex acts as a selective immobilised metal affinity adsorbent for phosphoproteins. [Muszyfiska et al. Biochemistry 25 6850 1986.]
Check Digit Verification of cas no
The CAS Registry Mumber 9001-78-9 includes 7 digits separated into 3 groups by hyphens. The first part of the number,starting from the left, has 4 digits, 9,0,0 and 1 respectively; the second part has 2 digits, 7 and 8 respectively.
Calculate Digit Verification of CAS Registry Number 9001-78:
(6*9)+(5*0)+(4*0)+(3*1)+(2*7)+(1*8)=79
79 % 10 = 9
So 9001-78-9 is a valid CAS Registry Number.
