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Proteinase K

AR0056

5 ml

Enabled

Proteinase K

Western Blotting Reagents, Sample Preparation

No

Boster’s Proteinase K is a Tris-HCl buffered concentrated enzyme stock solution that is to be used as assistant pre-treatment reagent in Western blot and IHC assay procedures.

Boster’s Proteinase K is a Tris-HCl buffered concentrated enzyme stock solution that is to be used as assistant pre-treatment reagent in Western blot and IHC assay procedures.

Overview Physical State Liquid Pack Size 5mL Form Supplied 1:100 Concentrated stock solution of Proteinase K in Tris-HCl buffer Content 10 mg/ml Proteinase K, 50mM Tris-HCl (pH 7.5), 150mM NaCl Reagent Type Assistant reagent Enzyme Concentration 10 mg/ml Recommended working concentration 50-100µg/ml for protein removal and enzyme inactivation; up to 2mg/ml for tissue treatment pH 7.5-9.0 Storage Store at -20 C for one year Equivalent Abcam (Product No. ab64220) Cite This Product Proteinase K (Boster Biological Technology, Pleasanton CA, USA, Catalog # AR0056) Precautions FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC AND CLINICAL USE Assay Principle . Proteinase K is a Tris-HCl buffered concentrated enzyme stock solution that is to be used as assistant pre-treatment reagent in Western blot and IHC assay procedures for general protein digestion in tissue lysates during sample preparation, for eliminating comigrating protein antigens prior to SDS-PAGE, as well as for proteolytic antigen retrieval prior to antibody staining in IHC. Application . •Proteolytic induced epitope retrieval (PIER); . •Proteolytic inactivation of endonucleases during DNA/RNA isolation and tissue sec•tions preparation for in-situ hybridization; . •Determination of enzyme localization; . •General protein digestion; . •Protein modification; . •Improve cloning efficiency of PCR products Biochemical Information CAS 39450-01-6 EC Numbers 3.4.21.64 Molecular Weight 28.93 Isoelectric Point 8.9 Enzyme Specificity Broad-spectrum serine protease Cleavage Site Peptide bonds adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups Active Site Active-site catalytic triad Asp39-His69-Ser224 Enzyme activity & Stability Ca2+ improve stability Stable in pH range 4–12; pH optimum 7.5 - 9.0 Stable and active in T range 37 - 65 °C; max activity at 50–60 °C Stable and active under denaturing conditions: SDS; EDTA; urea; citrate; Triton X-100; Tween 20; Guanidinium chloride; Guanidinium thiocyanate; Sarkosyl; iodoacetic acid; TLCK; TPCK Inhibition T > 65 °C; DIFP; PMSF; AEBSF; Hg2+ Enzyme Sources IntEnz BRENDA ExPASy Enzyme activity in commonly used buffers . (Measured under the following conditions: pH = 8.0, 50 °C, 1.25 µg/ml protease K, 15 min incubation) Buffer Proteinase K activity (%) 30 mM Tris·Cl 100 30 mM Tris•Cl; 30 mM EDTA; 5% Tween 20; 0.5% Triton X-100; 800 mM GuHCl 313 36 mM Tris•Cl; 36 mM EDTA; 5% Tween 20; 0.36% Triton X-100; 735 mM GuHCl 301 10 mM Tris·Cl; 25 mM EDTA; 100 mM NaCl; 0.5% SDS 128 10 mM Tris•Cl; 100 mM EDTA; 20 mM NaCl; 1% Sarkosyl 74 10 mM Tris•Cl; 50 mM KCl; 1.5 mM MgCl2; 0.45% Tween 20; 0.5% Triton X-100 106 10 mM Tris·Cl; 100 mM EDTA; 0.5% SDS 120 30 mM Tris·Cl; 10 mM EDTA; 1% SDS 203 Background . Proteinase K is a is a subtilisin-related endolytic non-specific serine protease with broad cleavage specificity on native and denatured proteins that cleaves ester and peptide bonds at the carboxylic sides of N-substituted hydrophobic aliphatic and aromatic amino acids. General features and behavior: It has high activity and remains stable across a wide range of pH (4.0-12.5) and temperature (25°C to 65°C) conditions and is suited to short digestion times. It remains active in the presence of various detergents and denaturants, and is even stimulated when up to either 2% SDS, 4 M urea, 3 M Guanidinium chloride, or 1 M Guanidinium thiocyanate are included in the reaction, making the substrate cleavage sites more accessible. Calcium ions, though contributing to Proteinase K stability, are not essential to the function of the enzyme, therefore it is also active in buffers containing metal chelating agents such as EDTA and may be used to inactivate calcium-dependent nucleases. It is also not inhibited by sulfhydryl reagents, trypsin and chymotrypsin inhibitors, or by serine protease inhibitors like Nalpha-Tosyl-Lys Chloromethyl Ketone (TLCK) and Nalpha-Tosyl-Phe Chloromethyl Ketone (TPCK). Proteinase K is however inhibited by temperatures above 65 °C, trichloroacetic acid (TCA) or the serine protease-inhibitors diisopropylfluorophosphate (DIFP), phenylmethylsulfonyl fluoride (PMSF), or 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF). Applications: Proteinase K is widely used to digest endogenous DNases and RNases and remove protein contaminations during nucleic acid isolation and purification from cell lysates and for preparation of tissue sections for in situ hybridization. Proteinase K is suitable for isolating PCR and RT-PCR templates. It has been used to remove endotoxins bound to cationic proteins such as lysozyme and ribonuclease. It has been reported useful for determination of enzyme localization on membranes, as well as for mitochondria isolation. In IHC it is used for treatment of paraffin embedded tissue sections to retrieve masked antigen binding sites for antibody labeling. In Western blot it is used as assistant reagent for general protein digestion during sample preparations.

5 ml

No

Store at -20 C for one year.

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