Description
Collagenase NB 5 is a crude collagenase that contains collagenolytic and additional enzymatic activities including clostripain and neutral protease. The balanced ratio of these activities ensures gentle and efficient tissue dissociation.Collagenase NB 5 is applicable to tissue dissociation such as isolation of human and rodent hepatocytes, chondrocytes, neuronal and endothelial cells, fibroblasts, cells from adipose tissue (e.g. ADSCs), stem cells and rodent osteoblasts.ApplicationCollagenase NB 5 is suitable for dissociation of a broad variety of tissue types. SpecificationsContains class I and class II collagenase (1) and a balanced ratio of proteolytic side activities.Activity (Wünsch): ≥ 0.10 U/mgMicrobiological examination: Sterility, absence of ClostridiaSterile according to Ph. Eur.(Clostridiopeptidase A)EC 3.4.24.3 • Mr ca. 70 000 - 120 000 (collagenases) • CAS [9001-12-1]References and DefinitionsUnit definition: 1 U according to Wünsch (2) catalyzes the hydrolysis of 1 µmole 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucylglycyl-L-prolyl-D-arginine per minute at 25 °C, pH 7.1.References1. Bond, M.D. & van Wart, H.E. (1984) Biochemistry 23, 3077-30912. Wünsch, E. & Heidrich, H.G. (1963) Hoppe-Seyler's Z. Physiol. Chem. 333, 149-51
Collagenases from Clostridium histolyticum are proteolytic enzymes that cleave peptide bonds in the triple helical collagen molecules of human or animal tissue in situ. For this reason collagenases are widely used for isolation of various cell types by tissue dissociation.Collagenase NB 5 Sterile Grade is sterile according to European Pharmacopoeia. Collagenase NB 5 Sterile Grade is a crude collagenase that contains collagenolytic and additional enzymatic activities including clostripain and neutral protease. The balanced ratio of these activities ensures gentle and efficient tissue dissociation. SpecificationCollagenase activity: ≥0.10 U/mg (PZ acc. to Wünsch) Sterility: must comply Application Collagenase NB 5 Sterile Grade is suitable for dissociation of a broad variety of tissue types.Storage conditionsCollagenase NB 5 Sterile Grade is available as a lyophilized powder.It should be stored at +2 to +8℃ in a dry environment. Under these conditions the product is stable until the minimum shelf life stated on the certificate of analysis if repeated opening and closing of the vial is avoided. For storage of solutions please refer to “Stock solution”. Instructions for use: General Collagenase NB 5 Sterile Grade is suitable for isolation of a broad variety of cells from human or animal tissues. Tissue types include adipose tissue, cartilage, skin, placenta, and umbilical cord tissue. It can also be applied in cell culture for passaging, e.g. of embryonic stem cells. Tissue dissociationRecommended starting concentrations for selected applications: Adipose tissue (human, rodent): 0.2 – 0.3 PZ U/ml Cartilage (human, rodent): 0.3 – 0.4 PZ U/ml In general, the appropriate collagenase concentration depends on tissue type and origin as well as on the isolation procedure.Collagenase activity is at an optimum at 37℃ and pH 7.4.Stock solution Collagenase NB 5 Sterile Grade dissolves at a concentration of up to 150 mg/ml in all buffers which are commonly used for cell isolation. The enzyme solution must be constantly stored on ice. Since collagenase and some of the secondary proteases depend on calcium, it is recommended to use a buffer with ≥ 2 mM Ca²⁺. Absolutely no calcium chelating agents (e.g. EDTA) should be present at all. Reconstituted Collagenase NB 5 Sterile Grade can be aliquoted and stored at -20 °C. Aliquots are stable for 1 year if repeated freezing and thawing is avoided. Collagenase NB 5 Sterile Grade is sterile according to Ph. Eur. Therefore, 0.22 µm filtration is not necessary if sterile equipment and buffers are used. If 0.22 µm filtration is required, filters with low protein-binding properties (e.g. cellulose acetate, PVDF, or PES) are recommended.Working solutionTo prepare a working solution, the stock solution is diluted with buffer to achieve the required collagenase concentration. The working solution must be constantly stored on ice until use.Inactivation and inhibitors The dissociation process can be reduced, e.g. by cooling down or dilution of the enzyme solution.Collagenase is reversibly inactivated at high pH values and irreversibly inactivated at low pH values. Inhibitors of collagenase include cysteine or chelating agents like EDTA. Important note Collagenase NB 5 Sterile Grade is intended for research use only