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| Product Name | Collagenase Type V |
| Catalog No. | RESK-0229 |
| Description | Collagenase is a protease, an endopeptidase, that specifically recognizes the Pro-X-Gly-Pro sequence (which occurs at high frequency in collagen and is rarely found in other proteins) and cleaves the peptide bond between the neutral amino acid (X) and glycine (Gly) in this sequence. Although many proteases can hydrolyze single-stranded, denatured collagen polypeptides, collagenase is the only protease capable of degrading native collagen fibers with a triple superhelical structure, which are widely present in connective tissue. This product is derived from Clostridium histolyticum and is a crude enzyme preparation. In addition to collagenase (more precisely termed clostridiopeptidase A), which degrades native collagen and reticular fibers, it also contains other proteases, polysaccharidases, and lipases that effectively hydrolyze other proteins, polysaccharides, and lipids in the extracellular matrix of connective and epithelial tissues, making it highly suitable for tissue digestion. Type V collagenase has higher collagenase and caseinase activities and a lower tryptic level to reduce damage to cell membrane proteins, making it suitable for the isolation of pancreatic islet cells and connective tissue cells. |
| Intended Use | Suitable for the isolation of pancreatic islet cells and connective tissue cells. Type V collagenase contains higher collagenase and caseinase activities and a lower tryptic level to reduce damage to cell membrane proteins. |
| Principle / Technology | Collagenase specifically recognizes the Pro-X-Gly-Pro sequence and cleaves the peptide bond between the neutral amino acid (X) and glycine (Gly); it is the only protease that degrades native triple superhelical collagen fibers in connective tissue. |
| Sample Type | Pancreatic islet tissue and connective tissue; also applicable to general tissue dissociation, organ perfusion, and cartilage digestion. |
| Performance Range / Specifications | Activity: ≥ 125 CDU/mg solid (CDU = collagen digestion units). Molecular weight: 68–130 kDa. Activators: Ca2+, Zn2+. Inhibitors: EDTA, EGTA, cysteine, histidine, 2-mercaptoethanol, o-phenanthroline, DTT, Hg2+, Pb2+, Cd2+, Cu2+, Zn2+; not inhibited by DFP or serum. |
| Specificity | Specifically recognizes the Pro-X-Gly-Pro sequence (frequent in collagen, rare in other proteins) and cleaves the peptide bond between the neutral amino acid (X) and glycine (Gly); uniquely degrades native triple superhelical collagen fibers. |
| Reaction Conditions / Protocol | Preparation of collagenase stock solution: Add 1 mL of HBSS (Hank's Balanced Salt Solution, containing Ca2+ and Mg2+) to each 100 mg vial of collagenase and gently vortex to fully dissolve, preparing a 100 mg/mL (100×) stock solution. Sterilize by filtration through a low-protein-binding 0.22 μm filter, aliquot into small portions, and store frozen at -20 °C protected from light. Thaw on ice before use and avoid repeated freeze-thaw cycles. Common working concentrations: 0.5–2.5 mg/mL for tissue and cell dispersion; 1–2 mg/mL for cartilage digestion. Determine the optimal working concentration according to specific experimental conditions or relevant literature. Tissue dissociation: 1) Cut tissue into 3–4 mm pieces using a sterile scalpel or scissors. 2) Wash the tissue pieces several times with HBSS containing Ca2+ and Mg2+. 3) Add sufficient HBSS (with Ca2+ and Mg2+) to submerge the tissue pieces and add collagenase to the desired working concentration. 4) Incubate at 37 °C for 4–18 h. Using a horizontal shaker during digestion and supplementing with 3 mM CaCl2 can improve digestion efficiency. 5) Collect dispersed cells using a stainless steel or nylon mesh; incompletely dissociated tissue can be further incubated with fresh collagenase working solution at 37 °C. 6) Wash the collected cells several times with collagenase-free HBSS. 7) Resuspend cells in cell culture medium and determine viable cell density using an automated cell counter or other methods. 8) Seed the cells on culture dishes with appropriate cell culture medium. Organ perfusion: 1) Add collagenase to HBSS (with Ca2+ and Mg2+) pre-warmed to 37 °C; adding 3 mM CaCl2 helps improve isolation efficiency. 2) Perfuse the organ with the collagenase working solution at an optimized rate. 3) Pass the recovered perfusate through a stainless steel or nylon mesh to separate dissociated cells or small tissue fragments from larger clumps; insufficiently dissociated tissue requires further incubation with fresh collagenase working solution at 37 °C. 4) Wash the collected cells several times with collagenase-free HBSS. 5) Resuspend cells in cell culture medium and determine viable cell density. 6) Seed the cells on culture dishes with appropriate cell culture medium. |
| Components / Formulation | Crude enzyme preparation containing collagenase (clostridiopeptidase A) plus other proteases, polysaccharidases, and lipases that hydrolyze proteins, polysaccharides, and lipids in the extracellular matrix of connective and epithelial tissues. |
| Storage Conditions | Store at 2–8 °C protected from light. Stock solutions should be stored frozen at -25 to -15 °C protected from light. |
| Shelf Life | 2 years when stored at 2–8 °C protected from light. |
| Package Specifications | 100 mg |
| Product Form | Lyophilized/solid powder, 100 mg per vial; reconstitute to prepare stock solution. |
| Activity / Unit Definition | ≥ 125 CDU/mg solid (CDU = collagen digestion units). One unit of enzyme activity is defined as the amount of enzyme that hydrolyzes collagen to produce an equivalent of 1 μM L-leucine within 5 hours at 37 °C, pH 7.5. |
| Molecular Weight | 68–130 kDa |
| Source / Origin | Derived from Clostridium histolyticum. |
| pH Range / Optimal pH | pH 7.5 (unit definition conditions: 37 °C, pH 7.5). |
| Expiration Date / Stability | 2 years at 2–8 °C protected from light; prepared stock solution should be stored at -25 to -15 °C protected from light, thawed on ice before use, and protected from repeated freeze-thaw cycles. |
| Recommended Buffer System | HBSS (Hank's Balanced Salt Solution) containing Ca2+ and Mg2+; supplementing the digestion with 3 mM CaCl2 improves digestion efficiency. HBSS in the buffer serves to enhance enzymatic digestion efficiency. |
| Application Notes / Precautions | Avoid repeated freeze-thaw cycles of the stock solution; thaw on ice before use. Tissue pieces can be digested repeatedly with collagenase: dispersed cells can be collected via stainless steel or nylon mesh, and incompletely dissociated tissue can be further incubated with fresh collagenase working solution at 37 °C. Activators: Ca2+, Zn2+; Inhibitors: EDTA, EGTA, cysteine, histidine, 2-mercaptoethanol, o-phenanthroline, DTT, Hg2+, Pb2+, Cd2+, Cu2+, Zn2+; not inhibited by DFP or serum. |
| Recognition Site | Pro-X-Gly-Pro sequence; cleaves the peptide bond between the neutral amino acid (X) and glycine (Gly). |
For research use only, not for clinical use.
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