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| Product Name | Trypsin 1:250 (Powder) |
| Catalog No. | RESK-0251 |
| Description | Trypsin is a serine protease commonly found in the digestive system of vertebrates. It is secreted by the pancreas as the inactive zymogen trypsinogen and hydrolyzes proteins by cleaving peptide chains mainly at the carboxyl side of lysine or arginine residues (except when followed by proline). Trypsin consists of two subunits: an alpha subunit (composed of 2 polypeptide chains) and a beta subunit (composed of 1 polypeptide chain). This product is a lyophilized trypsin powder derived from porcine pancreas, treated by gamma irradiation to inactivate viruses, with an enzymatic activity of ≥250 units/mg. It is widely used for cell passaging to detach adherent cells from culture vessels and prepare single-cell suspensions, at a typical working concentration of 0.25–5% (w/v). Trypsin activity can be inhibited by: 1) Organophosphorus compounds, such as diisopropyl fluorophosphate; 2) Natural trypsin inhibitors from pancreas, soybean, lima bean, and egg white; 3) Silver ions; 4) Specific protease inhibitors such as AEBSF, antipain, aprotinin, DFP, leupeptin, PMSF, and TLCK. |
| Intended Use | For routine cell culture passaging and primary tissue dissociation; detachment of adherent cells from culture vessels to prepare single-cell suspensions. For research use only. |
| Principle / Technology | Serine protease that cleaves peptide bonds at the carboxyl side of lysine and arginine residues, thereby hydrolyzing extracellular and adhesion proteins to release adherent cells from the culture surface. |
| Sample Type | Adherent cultured cells; primary tissues |
| Performance Range / Specifications | Appearance: white to off-white powder Specific activity: >250 U/mg Molecular weight: 23.8 kDa Extinction coefficient: E1%280 = 14.3 Isoelectric point: pH 10.5 Solubility: soluble in water; practically insoluble in ethanol and glycerol CAS No.: 9002-07-7 |
| Specificity | Cleaves peptide chains mainly at the carboxyl side of lysine or arginine residues, except when the residue is immediately followed by proline. |
| Reaction Conditions / Protocol | 1. Preparation of 0.25% trypsin stock solution Dissolve 0.25 g of trypsin powder in 100 mL of HBSS (without calcium and magnesium) and adjust the pH to 7.4–7.6. The resulting 0.25% trypsin solution can be stored at 4 °C for short-term use (relatively stable for 3 months); aliquoting and freezing is recommended for long-term storage. A small amount of precipitate may appear after thawing, which is normal and does not affect performance. The lyophilized powder can also be dissolved in other calcium/magnesium-free balanced salt solutions such as PBS. Cell digestion can be performed with trypsin solution alone, but trypsin-EDTA solution is commonly used because EDTA, a divalent metal ion chelator, chelates calcium and magnesium ions, reduces intercellular adhesion, and thereby enhances trypsin activity. 2. Preparation of trypsin-EDTA solution (0.25% w/v) Balanced salt solution without Ca2+, Mg2+ and phenol red: to 500 mL Trypsin: 1.25 g EDTA: 0.10 g 3. Digestion of adherent cells Method 1: 1) Remove the culture medium and wash the adherent cell monolayer with Ca2+/Mg2+-free buffer until residual serum is removed; aspirate the salt solution. 2) Add an appropriate volume of trypsin-EDTA solution to fully cover the cells. 3) Tilt the plate, aspirate the trypsin-EDTA solution, and rinse the cells by pipetting several times; do not over-digest. 4) Incubate at room temperature until the cells detach, monitoring under a microscope to avoid over-digestion and cell damage. 5) Add 10 mL of culture medium and gently pipette repeatedly to dislodge the cells, avoiding bubble formation. 6) Proceed to downstream steps or cryopreserve the cells. Method 2 (using a 25 cm2 T-flask as an example): 1) Aseptically aspirate the medium from the flask. 2) Add 3 mL of ice-cold trypsin-EDTA solution (formulation as above). 3) Incubate for 30 s (or longer if needed). Observe under a low-power microscope; when some cells begin to round up and detach, aspirate the trypsin solution and continue incubation until the cells fully detach. 4) Add 10 mL of fresh MEM medium quickly to help detach the cells; using the same pipette tip, gently rinse the cells several times, mix, and transfer 5 mL to a new flask. 5) Incubate under culture conditions. |
| Components / Formulation | Lyophilized trypsin powder derived from porcine pancreas; gamma-irradiated to inactivate viruses. |
| Storage Conditions | Store the lyophilized powder dry at 2–8 °C. |
| Shelf Life | 2 years |
| Package Specifications | 25 g |
| Product Form | Lyophilized powder (white to off-white) |
| Purity | Gamma-irradiated to inactivate viruses |
| Activity / Unit Definition | Specific activity: >250 U/mg. Unit definition: one unit is the activity causing a change in absorbance of 0.003/minute at 253 nm. |
| Molecular Weight | 23.8 kDa |
| Source / Origin | Porcine pancreas |
| pH Range / Optimal pH | Working solution pH 7.4–7.6; isoelectric point pH 10.5 |
| Shipping Conditions | Shipped with cold chain recommended; store at 2–8 °C upon receipt. |
| Expiration Date / Stability | 2 years when stored dry at 2–8 °C. The prepared 0.25% solution is relatively stable for 3 months at 4 °C; aliquot and freeze for long-term storage. |
| Regulatory / Compliance | For research use only. |
| Compatibility | Compatible with calcium/magnesium-free balanced salt solutions such as HBSS and PBS; activity is enhanced by EDTA and inhibited by serum, organophosphorus compounds, natural trypsin inhibitors, silver ions, and specific protease inhibitors (AEBSF, antipain, aprotinin, DFP, leupeptin, PMSF, TLCK). |
| Recommended Buffer System | HBSS or PBS without calcium and magnesium; typically used as trypsin-EDTA solution (0.25% trypsin, 0.02% EDTA). |
| Application Notes / Precautions | Digestion time depends on cell type, cell density, serum concentration used, trypsin activity, and the duration of cell culture prior to digestion. Avoid over-digestion to prevent trypsin-mediated cell damage. Handle carefully and avoid cross-contamination. A small amount of precipitate after thawing is normal and does not affect performance. |
| Recognition Site | Carboxyl side of lysine and arginine residues (except when followed by proline) |
For research use only, not for clinical use.
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