Proteinase K Powder (NGS)

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PREPARATION and SPECIFICATION

Appearance White amorphous powder, lyophilized
Activity ≥40 U/mg
Nucleic acid residue ≤5pg/mg
DNase None detected
RNase None detected

PROPERTIES

EC number 3.4.21.64  
Molecular weight 29 kDa (SDS-PAGE)  
Isoelectric point 7.81  
Optimum pH 7.5-12.0 Fig. 1
Optimum temperature 65 ℃ Fig. 2
pH stability pH 4.5-12.5 (25 ℃, 16 h) Fig. 3
Thermal stability Below 50 ℃ (pH 8.0, 30 min) Fig. 4
Activators SDS, urea  
Inhibitors Diisopropyl fluorophosphate;
phenylmethylsulfonyl fluoride
 

Applications

Genetic diagnostic kit; RNA and DNA extraction kits; Extraction of non-protein components from tissues, degradation of protein impurities, such as DNA vaccines and preparation of heparin; Preparation of chromosome DNA by pulsed electrophoresis; Western blot; Enzymatic glycosylated albumin reagents in vitro diagnosis.

Precautions

Wear protective gloves and goggles when using or weighing, and keep well ventilated after use. This product may cause skin allergic reaction. Cause serious eye irritation. If inhaled, it may cause allergy or asthma symptoms or dyspnea. May cause respiratory irritation.

ASSAY

Unit definition

One unit (U) is defined as the amount of enzyme required to hydrolyze casein to produce 1 μmol tyrosine per minute under the following conditions.

Reagents preparation

Reagent I: 1 g milk casein was dissolved in 50 ml of 0.1 M sodium phosphate solution (pH 8.0), incubated in 65-70 ℃ water for 15 min, stirred and dissolved, cooled by water, adjusted by sodium hydroxide to pH 8.0, and fixed volume 100ml.

Reagent II: 0.1 M trichloroacetic acid, 0.2 M sodium acetate, 0.3 M acetic acid.

Reagent III: 0.4 M Na2CO3 solution.

Reagent IV: Forint reagent diluted with pure water 5 times.

Reagent V: enzyme diluent: 0.1 M sodium phosphate solution (pH 8.0).

Reagent VI: tyrosine solution: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml tyrosine dissolved with 0.2 M HCl.

Procedure

0.5 ml of reagent I is pre-warmed to 37℃, add 0.5 ml of enzyme solution, mix well, and incubate at 37℃ for 10 min. Add 1 ml of reagent II to stop the reaction, mix well, and continue incubation for 30 min. Centrifuge the reaction solution.

Take 0.5 ml supernatant, add 2.5 ml reagent III, 0.5 ml reagent IV, mix well and incubate at 37℃ for 30 min. OD at 660 nm was determined as OD1; blank control group: 0.5 ml reagent V is used to replace enzyme solution to determine OD at 660 nm as OD2, △ OD=OD1-OD2.

L-tyrosine standard curve: 0.5 mL different concentration L-tyrosine solution, 2.5mL Reagent III, 0.5 mL Reagent IV in 5mL centrifuge tube, incubate in 37℃ for 30min, detect for OD at 660 nm for different concentration of L-tyrosine, then obtained the standard curve Y=kX+b, where Y is the L-tyrosine concentration, X is OD at 600 nm.