P5568

Proteinasi K

≥500 units/mL, buffered aqueous glycerol solution

• Sinonimo/i: Endopeptidasi K
• Numero CAS: 39450-01-6
• UNSPSC Code: 12352204
• eCl@ss: 32160410
• NACRES: NA.54
• Numero CE: 3.4.21.64 (BRENDA, IUBMB)
• MDL number: MFCD00132129
• Biological source: microbial (T.album; T. ALBUM)
• Concentration: ≥10 mg/mL, ≥500 units/mL

Proprietà

• biological source: microbial (T.album; T. ALBUM)
• Quality Segment: 200
• form: buffered aqueous glycerol solution
• mol wt: 28.93 kDa
• concentration: ≥10 mg/mL, ≥500 units/mL
• technique(s): DNA extraction: suitable
• storage temp.: 2-8°C

Descrizione

General description

Proteinase K, an extracellular endopeptidase is synthesized by the mold, Tritirachium album Limber. Proteinase K belongs to a new subfamily of the subtilisins. It is a 277 amino acid protein and is characterized with an unhydrolyzed protein chain and autolyzed polypeptide chains.

Application

Utile per l′inattivazione proteolitica delle nucleasi durante l′isolamento di DNA ed RNA.

Utile per l′inattivazione proteolitica delle nucleasi durante l′isolamento di DNA ed RNA.
Rimuove le endotossine che si legano alle proteine cationiche come lisozima e ribonucleasi A.
Segnalato come utile per l′isolamento dei mitocondri epatici, di lievito e di fagiolo mungo
Determinazione della localizzazione degli enzimi sulle membrane
Trattamento di sezioni di tessuto incluse in paraffina per esporre i siti di legame dell′antigene per la marcatura dell′anticorpo.
Digestione delle proteine da campioni di tessuto cerebrale per la ricerca sui prioni nell′encefalopatia spongiforme trasmissibile (EST).

Proteinase K from Tritirachium album has been used:

• to break down cardiac muscle during histopathology studies[1]
• during the digestion of HEK-293 cells[2]

The enzyme from Sigma has been used in the digestion of sealed cytosolic side out ER vesicles.[2] It has been used to deproteinize dissected brain and/or whole pupae sections of honey bee prior to in situ hybridisation. This was done during the study of neuropeptide Y-like signaling, and nutritionally-mediated gene expression and behaviour in the honey bee.[3]

Proteinase K from Tritirachium album has been used in in situ detection of DNA fragmentation and in proteolysis experiments to measure the structural flexibility of interleukin 1ra (IL-1ra).

Biochem/physiol Actions

La proteinasi K è una serin proteasi stabile e molto reattiva. Le evidenze emerse da studi sulla struttura cristallina e molecolare indicano che l′enzima appartiene alla famiglia delle subtilisine con una triade catalitica nel sito attivo (Asp³⁹-His⁶⁹-Ser²²⁴). È stabile in un vasto assortimento di ambienti: pH, sali tampone, detergenti (SDS) e temperatura. In presenza di SDS 0,1-0,5%, la proteinasi K conserva la sua attività e digerisce una serie di proteine e nucleasi nelle preparazioni di DNA senza compromettere l′integrità del DNA isolato.

Proteinase K has a broad specificity and degrades many proteins even in the native state. It mainly cleaves the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha-amino groups.[4][5] The optimum pH is between 7.5-9.0[4] and the isoelectric point is 8.9.[4] Ca²⁺ (1-5 mM) is required for activation. Proteinase K is inhibited by DIFP or PMSF.

Physical form

Solution in 40% (v/v) glycerol containing 10 mM Tris-HCl, pH 7.5, with 1 mM calcium acetate.

Preparation Note

Proteinase K in solution is stable over a pH range of 4.0-12.5 (optimum pH 8.0), and is also stable over the temperature range of 25°C to 65°C during use. At pH 8.0, solutions will be stable for at least 12 months at 4°C. At pH 4-11.5, solutions containing Ca²⁺ (1-6 mM) are expected to be stable for several weeks. An 80% ammonium sulfate suspension stored at 4°C is stable for at least 12 months.[4]

Other Notes

One unit will hydrolyze urea-denatured hemoglobin to produce color equivalent to 1.0 μmole of tyrosine per min at pH 7.5 at 37 °C (color by Folin-Ciocalteu reagent).

Informazioni sulla sicurezza

• pictograms: GHS08
• signalword: Danger
• hcodes: H334
• pcodes: P261 - P284 - P501
• Hazard Classifications: Resp. Sens. 1
• Classe di stoccaggio: 10 - Combustible liquids
• wgk: WGK 1
• flash_point_f: Not applicable
• flash_point_c: Not applicable
• ppe: Eyeshields, Faceshields, Gloves, type ABEK (EN14387) respirator filter