Collagenase NB and Neutral Protease
For efficient tissue dissociation in vitro
Collagenases from Clostridium histolyticum are proteolytic enzymes that are able to cleave peptide bonds in the triple helical collagen molecule of human or animal tissue in situ.The collagenases are divided into class I and class II collagenase isoforms on the basis of their activities towards synthetic peptides. Both collagenase isoforms act on triple helical type I, II, III and IV collagens, but in slightly different modes of action.
GMP compliant Collagenase NB qualities for the isolation of cells for use in a clinical setting meet the highest possible safety specifications.
Product safety is ensured by means of extensive microbiological controls. In addition, the products’ viral and TSE safety is assured by means of careful selection of the starting materials and the specific production process.
At present, Collagenase NB has found highly promising applications in regenerative medicine and tissue engineering. Human cells are isolated and grown, if applicable, into tissue in the laboratory. The cells or tissue are suitable for human transplantation either for regeneration purposes or to replace damaged tissue.
Stability and Storage
Collagenase requires calcium ions both for full catalytic activity and binding to the collagen molecule. Collagenase NB enzymes are soluble in water and diluted salt solutions. The enzyme is reversibly inactivated at high pH values and irreversibly inactivated at low pH values.
Inhibitors of Collagenase include cysteine, EDTA, o-phenanthroline, 8-hydroxyquinoline-5-sulfonate, bipyridyl and 2,3-dimercaptopropanol. It is also inhibited by TRIS buffer above pH 7.5. Collagenase is not inhibited by diisopropylphosphorofluoridate.
All Collagenase NB products delivered as lyophilized powders can be stored at +2 to +8°C and remain stable without loss of activity for at least three years. Enzymes should be protected from moisture. Dissolved enzymes can be aliquoted and stored at -20°C for one year. Repeated freezing and thawing should be avoided.
Conversion rates of Collagenase activity units
Our Collagenase activity is measured in PZ units according to Wünsch.
1 PZ U catalyzes the hydrolysis of 1 µmol 4-phenylazobenzyl- oxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-arginine (PZ) per minute at 25 °C, pH 7.1. Collagenase activity can also be measured in FALGPA units, collagen digestion units (CDU, also known as Mandl units) or HP units. There are no exact conversion factors, but the following approximate conversion factors can be applied:
1 PZ U ~ 3.9 FALGPA U
1 PZ U ~ 1000 Mandl U or CDU
1 PZ U ~ 10 HP U
We recommend dosing Collagenase NB by activity (PZ units), not by weight. We guarantee a high lot-to-lot consistency; however, small variations may occur. Therefore dosing by activity ensures consistent results for your cell isolation process.
Collagenase is often used in combination with Neutral Protease for cell isolation. Neutral Protease is an enzyme that can catalyse the hydrolysis of peptide bonds in neutral, weakly acidic or weakly alkaline environment. Its optimal pH is between 6.0 and 7.5. Nordmark Neutral Protease NB is also derived from C. histolyticum.
For cell isolation Neutral Protease NB GMP Grade is often used in combination with purified Collagenase NB 1 GMP Grade (Cat. no. N0002937) or Collagenase NB 6 GMP Grade (Cat. no. N0002779) for applications which have to meet GMP requirements like tissue engineering and transplantation into humans.
Clostripain also known as Clostridiopeptidase B or Endoproteinase Arg-C is a proteinase derived from C. histolyticum that cleaves peptide bonds on the carboxyl side of arginine. This protein is a heterodimer composed of a heavy and a light chain that are held together via strong noncovalent forces. As a proteolytic enzyme it can be used for isolation of various cell types by tissue dissociation.
Clostripain exists in two different forms, an oxidized, native condition and a reduced activated form that differ in enzymatic activity. The native clostripain can be activated with reducing agents (e.g. DTT, cysteine) resulting in a higher activity of the enzyme.
Clostripain NB is often used in combination with Collagenase NB 1 Premium Grade (Cat. No. S1745503) and Neutral Protease NB (Cat. No. S3030112). Clostripain NB can also be used with Collagenase NB 4 Standard Grade (Cat. No. S1745401) or Collagenase NB 8 Broad Range (Cat. No. S1745601), as well.
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Highly purified Clostripain NB, in combination with Nordmark’s other purified collagenases and neutral proteases will boost your cell isolation procedure!