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| Product Name | Collagenase Type IV |
| Catalog No. | RESK-0225 |
| Description | Collagenase is a protease, specifically 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). Although many proteases can hydrolyze single-stranded, denatured collagen polypeptides, collagenase is the only protease capable of degrading native collagen fibers with a triple-helical supercoiled structure, which are widely present in connective tissue. This product is derived from Clostridium histolyticum and is a crude enzyme preparation that contains not only collagenase (more accurately termed clostridiopeptidase A), which degrades native collagen and reticular fibers, but also 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 IV collagenase contains low trypsin-like (tryptic) activity and is commonly used for the preparation of pancreatic islet cells or for cell preparation experiments in which receptor integrity must be maintained. This product is Type IV collagenase with an activity of >=125 CDU/mg solid (CDU = collagen digestion units) and is especially suitable for the digestion of pancreatic islet tissue. |
| Intended Use | For tissue dissociation and cell isolation; especially suitable for pancreatic islet tissue digestion and for cell preparation experiments requiring preservation of cell surface receptor integrity. |
| Principle / Technology | Endopeptidase that specifically recognizes the Pro-X-Gly-Pro sequence in collagen and cleaves the peptide bond between the neutral amino acid (X) and glycine (Gly); the only protease able to degrade native triple-helical collagen fibers in connective tissue. |
| Performance Range / Specifications | Activity: >=125 CDU/mg solid (CDU = collagen digestion units) Low trypsin-like (tryptic) activity Contains collagenase (clostridiopeptidase A) plus additional proteases, polysaccharidases, and lipases to aid tissue digestion |
| 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 dissolve completely, yielding a 100 mg/mL (100x) stock solution. Sterilize by filtration through a low protein-binding 0.22 um membrane, aliquot, and store frozen at -20 °C protected from light. Thaw on ice before use and avoid repeated freeze-thaw cycles. The commonly used concentration for tissue and cell dispersion is 0.5-2.5 mg/mL; for cartilage digestion, 1-2 mg/mL is commonly used. Determine the optimal working concentration according to specific experimental conditions or relevant literature. Tissue dissociation: 1) Mince tissue into 3-4 mm pieces with a sterile scalpel or scissors. 2) Wash tissue pieces several times with HBSS containing Ca2+ and Mg2+. 3) Add sufficient HBSS containing Ca2+ and Mg2+ to submerge the tissue pieces, and add collagenase to the required 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 through a stainless steel or nylon mesh; incompletely dissociated tissue can be further incubated at 37 °C with fresh collagenase working solution. 6) Wash collected cells several times with collagenase-free HBSS. 7) Resuspend cells in culture medium and determine viable cell density using an automated cell counter or other methods. 8) Seed cells onto culture dishes with appropriate culture medium. Organ perfusion: 1) Add collagenase to HBSS containing Ca2+ and Mg2+ pre-warmed to 37 °C; adding 3 mM CaCl2 helps improve isolation efficiency. 2) Perfuse the corresponding 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 should be further incubated with fresh collagenase working solution at 37 °C. 4) Wash collected cells several times with collagenase-free HBSS. 5) Resuspend cells in culture medium and determine viable cell density. 6) Seed cells onto culture dishes with appropriate culture medium. |
| Components / Formulation | Crude enzyme preparation from Clostridium histolyticum containing collagenase (clostridiopeptidase A) together with other proteases, polysaccharidases, and lipases. Supplied as lyophilized powder. |
| Storage Conditions | Store at 2-8 °C protected from light. Prepared stock solutions should be stored frozen at -25 to -15 °C protected from light; avoid repeated freeze-thaw cycles. |
| Shelf Life | 2 years when stored at 2-8 °C protected from light. |
| Package Specifications | 100 mg 1 g 5 g |
| Product Form | Lyophilized powder. |
| Purity | Crude enzyme preparation (contains collagenase plus additional proteases, polysaccharidases, and lipases). |
| 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 release peptides equivalent to 1 uM L-leucine within 5 h at 37 °C, pH 7.5. |
| Source / Origin | Clostridium histolyticum |
| pH Range / Optimal pH | pH 7.5 (unit definition assay condition) |
| Shipping Conditions | Standard shipping; store at 2-8 °C upon receipt. |
| Expiration Date / Stability | 2 years at 2-8 °C protected from light; stock solutions stable at -25 to -15 °C protected from light (avoid repeated freeze-thaw). |
| Regulatory / Compliance | For research use only. Not for use in diagnostic or therapeutic procedures. |
| Recommended Buffer System | HBSS (Hank's Balanced Salt Solution) containing Ca2+ and Mg2+; supplementing with 3 mM CaCl2 can improve digestion efficiency. |
| Application Notes / Precautions | Avoid repeated freeze-thaw cycles of stock solutions; thaw on ice before use. Filter-sterilize stock solutions using a low protein-binding 0.22 um membrane. Collagenase activity is Ca2+-dependent; maintain Ca2+ and Mg2+ in the digestion buffer. |
| 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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