Product Description
Product Name Proteinase K, Recombinant
Cat. No. & Specifications C12100,1g/10g/100g
CAS: 39450-01-6, E.C. 3.4.21.64
Synonyms: Peptidase K, Endoproteinase K, Endopeptidase K
Introduction
Proteinase K is a stable serine protease with broad substrate specificity. It degrades many proteins in the native state even in the presence of detergents. It is not activated by metal ions, chelating agents (for example, EDTA), sulfhydryl reagents, or by tripsin or chymotrypsin inhibitors. It is stable over a wide pH range (4-12.5), with optimal activity at pH 6.5-9.5. Activity can be stimulated by addition of denaturing agents (SDS and Urea). Rapid denaturation of the enzyme occurs at temperature above 70°C. Autolysis of the enzyme occurs increasingly at alkaline pH. However, Proteinase K is not completely inactivated by autolysis. Some enzyme fragments continue to maintain their complete proteolytic activity, even after extensive autolysis.
Proteinase K is frequently used in molecular biology applications to digest unwanted proteins, such as nucleases in DNA or RNA preparations from microorganisms, cultured cells, and plants. The enzyme is typically used at 50-200 ug/ml in nucleic acid preparations at pH 7.5-8.0 and 37~55oC. Incubation times vary from 30 minutes to 18 hours.
Reconstitute Proteinase K
Dissolve in 20 mM Tris HCl, pH 7.5, 10 mM calcium chloride, and 50% glycerol, then store at -20~8°C. When Store at 2-8°C, Glycerol is optional. Storage at -20°C in the absence of glycerol can lead to precipitation of the Proteinase K. Bacterial growth can occur in solutions stored at 2-8°C over extended periods of time. Ca2+ can serve as a stabilizer to suppress autolysis.
Storage and stability
Magen recommends storage at -20~8°C, When stored at 2~8°C, the product retains activity for at least 2 years.
Applications
DNA/RNA Isolation.
Odering information
| Cat.No. |
PK-1 |
PK-10 |
PK-100 |
| Proteinase K, Lyophilizate, >30 units/mg of protein |
1g |
10g |
100g |
| Cat.No. |
PBD-100 |
PBD-1000 |
| Buffer PBD (20mM Tris, pH 7.5, 10mM CaCl2, 50% glycerol, 0.1% Preservatives) |
100 ml |
1000 ml |
Typical experiments
1.Isolation of gDNA: Dissolve the lyophilized Proteinase K at 20mg/ml in Buffe PBD and use it with HiPure Universal DNA Kit to isolation DNA from: 200ul mammalian blood, 2 x 106 cultured mammalian cells,10~20 mg mammalian tissue/mouse tail. 20ul of the reconstituted proteinase K solution is enough.
2.Preparation of tissue sections for in situ hybridization: For some tissues, treatment of cytological sections with proteinase K will improve the likelihood that probes will reach cellular nucleic acids. The effectiveness of proteinase K treatment and the optimal concentration of proteinase K depend greatly on the kind of tissue and how it was fixed. For example, to treat blood vessel or myocardial tissue, Plenz et al (7) used the following concentrations of proteinase K: Cryosections: up to 2 g/ml; Paraffin-embedded sections: up to 20 g/ml; Methacrylate-embedded sections: up to 50 g/ml.
Special Quality Inspection
1. DNase: none detected
2. Endonuclease (nickase): none detected
3. RNase: none detected
4. DNA detection: 20mg Proteinase K were constituted by 2ml 0.5% SDS and incubate at 65oC for 30 minutes. Then Total DNA were isolated using HiPure Circulating DNA Kit (D3182). Total DNA were eluted by 50ul Buffer TE.
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