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| Product Name | Collagenase Type I |
| Catalog No. | RESK-0216 |
| Description | Collagenase is an endopeptidase that specifically recognizes the Pro-X-Gly-Pro sequence, which occurs at high frequency in collagen and rarely 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 distributed in connective tissue. This product is a crude enzyme preparation derived from Clostridium histolyticum. In addition to collagenase (more precisely clostridiopeptidase A), which degrades native collagen and reticular fibers, it contains other proteases, polysaccharidases, and lipases that efficiently hydrolyze additional proteins, polysaccharides, and lipids in the extracellular matrix of connective and epithelial tissues, making it highly suitable for tissue digestion. This product is Type I collagenase, with an activity of ≥250 U/mg solid and a balanced spectrum of enzymatic activities (collagenase, caseinase, clostripain, and tryptic activities). It is suitable for the dissociation of epithelial, liver, lung, fat, and adrenal tissues and cells. |
| Intended Use | For research use in tissue dissociation and primary cell preparation. Recommended for the isolation of cells from epithelial, liver, lung, fat, and adrenal tissues. |
| Principle / Technology | Type I collagenase provides a balanced profile of collagenase, caseinase, clostripain, and tryptic activities. Collagenase (clostridiopeptidase A) uniquely degrades native triple-helical collagen fibers by cleaving the Pro-X-Gly-Pro motif between the neutral amino acid (X) and glycine. Auxiliary proteases, polysaccharidases, and lipases in the crude preparation digest other extracellular matrix components, enabling efficient tissue disaggregation. |
| Sample Type | Animal tissues (epithelial, liver, lung, fat, adrenal) and cultured cells. |
| Performance Range / Specifications | Activity: ≥250 U/mg solid. Typical working concentration: 0.5–2.5 mg/mL for tissue and cell dispersion; 1–2 mg/mL for cartilage digestion. Recommended working concentration range: 0.05%–0.5% (w/v). |
| Reaction Conditions / Protocol | 1. Preparation of stock solution Add 1 mL of HBSS (Hank's Balanced Salt Solution) containing Ca2+ and Mg2+ to each 100 mg vial of collagenase and vortex gently to dissolve completely, yielding a 100 mg/mL (100×) 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. 2. Tissue dissociation a) Mince the tissue into 3–4 mm pieces with a sterile scalpel or scissors. b) Wash the tissue pieces several times with HBSS containing Ca2+ and Mg2+. c) Add sufficient HBSS (with Ca2+/Mg2+) to submerge the tissue and add collagenase to the desired working concentration. d) Incubate at 37 °C for 4–18 h. Digestion on a horizontal shaker and supplementation with 3 mM CaCl2 improve digestion efficiency. e) Collect dispersed cells through a stainless steel or nylon mesh; incompletely dissociated tissue can be further incubated with fresh collagenase working solution at 37 °C. f) Wash the collected cells several times with collagenase-free HBSS. g) Resuspend the cells in culture medium and determine viable cell density with an automated cell counter or another suitable method. h) Seed the cells onto culture dishes with appropriate medium. 3. Organ perfusion a) Add collagenase to HBSS (with Ca2+/Mg2+) prewarmed to 37 °C; an additional 3 mM CaCl2 helps improve isolation efficiency. b) Perfuse the organ with the collagenase working solution at an optimized flow rate. c) 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. d) Wash the collected cells several times with collagenase-free HBSS. e) Resuspend the cells in culture medium and determine viable cell density. f) Seed the cells onto culture dishes with appropriate medium. 4. Preparation of skin single-cell suspension a) Digest minced skin tissue in RPMI-1640 medium containing 2 mg/mL Collagenase Type I, 2.4 U/mL Dispase II, and 1 mg/mL DNase at 37 °C for 90 min. b) After digestion, wash with HBSS buffer containing 0.5% BSA and filter through a 40 um cell strainer to obtain a single-cell suspension. |
| Components / Formulation | Crude enzyme preparation containing collagenase (clostridiopeptidase A) together with other proteases, polysaccharidases, and lipases. Supplied as a lyophilized powder. |
| Storage Conditions | Store at 2–8 °C protected from light. Store the reconstituted stock solution frozen at -20 °C protected from light and avoid repeated freeze-thaw cycles. |
| Shelf Life | 2 years when stored at 2–8 °C protected from light. |
| Package Specifications | 100 mg 1 g |
| Product Form | Lyophilized powder |
| Activity / Unit Definition | ≥250 U/mg solid. One unit of enzyme activity is defined as the amount of enzyme that hydrolyzes collagen to release the equivalent of 1 uM L-leucine within 5 h at 37 °C, pH 7.5. |
| Source / Origin | Clostridium histolyticum |
| pH Range / Optimal pH | Enzyme activity unit is defined at pH 7.5. |
| Shipping Conditions | Shipped at room temperature. |
| Compatibility | Collagenase activity is not affected by serum used in cell culture. Activity is inhibited by chelators (EDTA, EGTA), heavy metal ions (Hg2+, Pb2+, Cd2+), and reagents such as cysteine, histidine, DTT, 2-mercaptoethanol, and o-phenanthroline. |
| Recommended Buffer System | HBSS (Hank's Balanced Salt Solution) containing Ca2+ and Mg2+; Earle's or other balanced salt solutions are also suitable for reconstitution. |
| Application Notes / Precautions | Collagenase is not affected by serum in culture medium. Inhibitors include EDTA, EGTA, heavy metal ions (Hg2+, Pb2+, Cd2+), cysteine, histidine, DTT, 2-mercaptoethanol, and o-phenanthroline. Dissolve in Hank's, Earle's, or other balanced salt solutions rather than PBS; recommended working concentration is 0.05%–0.5% (w/v). If digestion is poor, verify enzyme activity and calcium supply; set the Ca2+ concentration in the digestion solution to 5 mM, and store collagenase dry at low temperature with aliquoted frozen stock solutions. If cell death occurs, reduce protease exposure time or add albumin or heat-inactivated serum; maintain pH with a buffered solution such as HBSS and check/re-adjust pH regularly; if oxygen is limiting, aerate the digestion solution with sterile air. For crude tissue dissociation, crude collagenase preparations are appropriate; for studies of collagen structure and biosynthesis, use highly purified collagenase free of other proteolytic activities. For downstream flow cytometry, gentler collagenase types (Type III or IV) have less impact on cell surface antigens. |
| 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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