| Description | Enzymes extracted from mammalian testes (e.g., sheep testes) can hydrolyze mucopolysaccharides of the hyaluronic acid type. They may contain a suitable stabilizer. Potency: Minimum 1000 IU of hyaluronidase activity per milligram (of dry substance).ProductionAnimals used for producing hyaluronidase Enzymes extracted from mammalian testes (e.g., sheep testes) can hydrolyze mucopolysaccharides of the hyaluronic acid type. They may contain a suitable stabilizer. Potency: Minimum 1000 IU of hyaluronidase activity per milligram (of dry substance).ProductionAnimals used for producing hyaluronidase must meet the health requirements for animals intended for human consumption.CharacteristicsAppearance: White or yellowish-white, amorphous powder.Solubility: Soluble in water, almost insoluble in acetone and absolute ethanol.IdentificationA solution containing 100 IU of hyaluronidase in 1 mL of 9 g/L sodium chloride solution depolymerizes a 10 g/L sodium hyaluronate BRP solution at 20°C, resulting in a significant decrease in viscosity. Heating the hyaluronidase at 100°C for 30 minutes destroys this effect.Tests1.Appearance of Solution: The solution should be clear. Dissolve 0.10 g in water and dilute to 10 mL with the same solvent.2.pH: 4.5 to 7.5. Dissolve 30 mg in carbon dioxide-free water and dilute to 10 mL with the same solvent.3.Loss on Drying: Maximum 5.0%. Determine by drying 0.500 g at 60°C under a pressure not exceeding 670 Pa for 2 hours.4.Bacterial Endotoxins: ≤ 0.2 EU/IU.AssayThe activity of hyaluronidase is determined using a slope-ratio assay, by comparing the rate at which it hydrolyzes sodium hyaluronate BRP with the rate obtained using the International Standard or a reference preparation calibrated in International Units.Substrate SolutionIn a 25 mL conical flask, add 0.10 g of sodium hyaluronate BRP, then slowly add 20.0 mL of water at 4°C. The addition rate must be slow enough to allow the substrate particles to swell (approximately 5 minutes). Maintain at 4°C and stir for at least 12 hours. Store at 4°C and use within 4 days.For both the test solution and the reference solution, prepare the solutions and perform dilutions at 0°C to 4°C.Test Solution: Dissolve an appropriate amount of the substance in hyaluronidase diluent to obtain a solution containing 0.6 ± 0.3 IU of hyaluronidase per mL.Reference Solution: Dissolve an appropriate amount of hyaluronidase BRP in hyaluronidase diluent to obtain a solution containing 0.6 IU of hyaluronidase per mL.In a reaction vessel, mix 1.50 mL of phosphate buffer solution (pH 6.4) and 1.0 mL of the substrate solution, and equilibrate at 37 ± 0.1°C. At time t₀ = 0 (using the first timer), add 0.50 mL of the test solution containing E milligrams of the enzyme to be tested, mix well. Maintain the mixture at 37 ± 0.1°C using a suitable viscometer, record the flow time t using a second timer (with 0.1-second intervals), and perform multiple measurements over approximately 20 minutes (monitoring with the first timer). Use the following viscometer: microviscometer (DIN 51 562, Part 2), capillary type MII, with a viscometer constant of approximately 0.1 mm²/s².Repeat the above procedure using 0.50 mL of the reference solution containing hyaluronidase BRP. Calculate the viscosity ratio using the following expression:K = Viscometer constant (in mm²/s², indicated on the viscometer);t₂ = Flow time of the solution (in seconds);0.6915 = Kinematic viscosity of the buffer solution at 37°C (in mm²/s).Since the enzymatic reaction continues during the flow time measurement, the actual reaction time is equal to t₀ + t/2 (i.e., half of the flow time (t/2) is added to the initial measurement time t₀). Plot (ln η)⁻¹ as a function of the reaction time (t₀ + t/2) (in seconds); a linear relationship should be obtained. Calculate the slope (b) of the substance to be tested and the slope (bᵣ) of the reference preparation. Determine the specific activity in International Units per milligram using the following expression:A = Specific activity of hyaluronidase BRP (in International Units per milligram).Perform at least three complete sets of the procedure and calculate the average activity of the substance to be tested.StorageStore in a tightly closed container at a temperature of 2°C to 8°C. If the substance is sterile, the container should also be sterile and tamper-proof.
Hyaluronidase is present in high amount in testis as it aids fertilization of the mammalian egg. Mammalian hyaluronidase has a catalytic epidermal growth factor (EGF)-like domain and a C-terminal cysteine rich region.Application:Hyaluronidase has been used:as a component of enzyme mix for the isolation of sertoli and germ cells from seminiferous tubulesin the pre-treatment of deparaffinized adenomas sections for immunohistochemistryin the digestion of umbilical cord blood vessel for the generation of umbilical cord stromal stem cell lines... Read More | TEV Protease is the 241 amino acid (aa), 27 kDa catalytic domain of the nuclear inclusion a (NIa) protein encoded by the potyvirus, tobacco etch virus (TEV). It may be used in biotechnology to cleave affinity tags from recombinant proteins, either co-translationally orin vitrofollowing purification.TEV Protease is the 241 amino acid (aa), 27 kDa catalytic domain of the nuclear inclusion a (NIa) protein encoded by the potyvirus, tobacco etch virus (TEV). It may be used in biotechnology to cleave affinity tags from recombinant proteins, either co-translationally orin vitrofollowing purification. Its high specificity and activity at a wide range of pH and ionic strength make TEV Protease more versatile than many other proteases used for the same purpose. Unlike factor Xa, enteropeptidase or thrombin, TEV Protease has not been found to cleave at unintended sites, even when present at a high concentration. TEV Protease is a 3C-type protease that cleaves substrates with a consensus sequence of ENLYFQG. Cleavage occurs between Q and G. Since the final aa remains on the cleaved protein where it could potentially affect structure or function, substitution of a variety of aa have been tested. In order of efficiency, S, A, M, Y, D, N, E, K or L may be effectively used in place of G. Several of the remaining aa may also vary, giving a final consensus sequence of ExxYF(M)Q(E)/G(S, A or others) where aa in parenthesis are alternatives and x is any aa. The autocatalytic site of NIa at S2256 has been mutated to an N for improved stability of the protease.Tobacco Etch Virus Protease is a highly site-specific cysteine protease that is found in the tags from fusion proteins. The optimal temperature for cleavage is 30°C. It is recommended that the cleavage for each fusion protein be optimized by varying the amount of recombinant viral TEV protease, reaction time, or incubation temperature. It can be removed by Ni2+ affinity resin... Read More | Purity>95% (SDS-PAGE&HPLC) Endotoxin level<1.0 EU/µgFunctionBifunctional growth-modulating glycoprotein. Inhibits growth of several human carcinoma cells in culture and stimulates proliferation of human fibroblasts and certain other tumor cells | Purity:>95%, by SDS-PAGE visualized with Coomassie® Blue StainingDescription:Bcl-2 family proteins contribute to programmed cell death or apoptosis. It is a large protein family and all members contain at least one of four Bcl-2 homology domains. Certain members (Bcl-2, Bcl-XL and Mcl-1) arePurity:>95%, by SDS-PAGE visualized with Coomassie® Blue StainingDescription:Bcl-2 family proteins contribute to programmed cell death or apoptosis. It is a large protein family and all members contain at least one of four Bcl-2 homology domains. Certain members (Bcl-2, Bcl-XL and Mcl-1) are antiapoptotic, whilst others (Bax, Bak, Bok) are proapoptotic... Read More | Background：Tumor necrosis factor alpha (TNF-alpha ), also known as cachectin and TNFSF2, is the prototypic ligand of the TNF superfamily. It is a pleiotropic molecule that plays a central role in inflammation, immune system development, apoptosis, and lipid metabolism. Rat TNF-alpha consisitsBackground：Tumor necrosis factor alpha (TNF-alpha ), also known as cachectin and TNFSF2, is the prototypic ligand of the TNF superfamily. It is a pleiotropic molecule that plays a central role in inflammation, immune system development, apoptosis, and lipid metabolism. Rat TNF-alpha consisits of a 35 amino acid (aa) cytoplasmic domain, a 21 aa transmembrane segment, and a 179 aa extracellular domain (ECD). Within the ECD, rat TNF-alpha shares 94% aa sequence identity with mouse and 69%-76% with bovine, canine, cotton rat, equine, feline, human, porcine, and rhesus TNF-alpha. TNF-alpha is produced by a wide variety of immune, epithelial, endothelial, and tumor cells. TNF-alpha is assembled intracellularly to form a noncovalently linked homotrimer which is expressed on the cell surface. Cell surface TNF-alpha can induce the lysis of neighboring tumor cells and virus infected cells, and it can generate its own downstream cell signaling following ligation by soluble TNFR I. Shedding of membrane bound TNF-alpha by TACE/ADAM17 releases the bioactive cytokine, a 55 kDa soluble trimer of the TNF-alpha extracellular domain. TNF-alpha binds the ubiquitous 55-60 kDa TNF RI and the hematopoietic cell-restricted 80 kDa TNF RII, both of which are also expressed as homotrimers. Both type I and type II receptors bind TNF-alpha with comparable affinity, although only TNF RI contains a cytoplasmic death domain which triggers the activation of apoptosis. Soluble forms of both types of receptors are released and can neutralize the biological activity of TNF-alpha. Post-translational modificationsThe soluble form derives from the membrane form by proteolytic processing.The membrane form, but not the soluble form, is phosphorylated on serine residues.Dephosphorylation of the membrane form occurs by binding to soluble TNFRSF1A/TNFR1.O-glycosylated; glycans contain galactose, N-acetylgalactosamine and N-acetylneuraminic acid... Read More |