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SKU AR0056
Pack size 5 mL
Form Solution (liquid)
Applications Proteolytic induced epitope retrieval (PIER); Proteolytic inactivation of endonucleases during DNA/RNA isolation and tissue sections preparation for in-situ hybridization; Determination of enzyme localization; General protein digestion; Protein modification; Improve cloning efficiency of PCR products
Price: $50.00
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Overview

Product Name Proteinase K
Synonyms Protease K, Endopeptidase K, Tritirachium Alkaline Proteinase
Description 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 sections preparation for in-situ hybridization; Determination of enzyme localization; General protein digestion; Protein modification; Improve cloning efficiency of PCR products
Pack Size 5 mL
Reagent Type Assistant reagent

Properties

Content 1 mg/ml Proteinase K, 50mM Tris-HCl (pH 7.5), 150mM NaCl
Form Supplied 1:100 Concentrated stock solution of Proteinase K in Tris-HCl buffer
Physical State Liquid
Pack Size 5ml
Enzyme Concentration 1 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
Precautions FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC AND CLINICAL USE

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 Nα-Tosyl-Lys Chloromethyl Ketone (TLCK) and Nα-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.

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Proteinase K
Proteinase K (AR0056)
5 mL per pack, Solution (liquid)
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Publications

Cheng O, Li Z, Han Y, Jiang Q, Yan Y, Cheng K. Brain Res. 2012 Aug 27;1470:111-8. Doi: 10.1016/J.Brainres.2012.06.026. Epub 2012 Jul 11. Baicalin Improved The Spatial Learning Ability Of Global Ischemia/Reperfusion Rats By Reducing Hippocampal Apo...