| Description | ApplicationOxidized glycosylated amino acids, used in the development and mass preparation of enzymatic glycosylated hemoglobin reagents.Enzymatic propertiesSource: MicroorganismEnzymology Committee Number: EC1.5.3Molecular weight: 60 kDa (SDS-PAGE)Isoelectric point: 6.4Km value: 4.0×10-3M (ApplicationOxidized glycosylated amino acids, used in the development and mass preparation of enzymatic glycosylated hemoglobin reagents.Enzymatic propertiesSource: MicroorganismEnzymology Committee Number: EC1.5.3Molecular weight: 60 kDa (SDS-PAGE)Isoelectric point: 6.4Km value: 4.0×10-3M (fructosyl-Val-His)Inhibitors: Hg²⁺, Pb²⁺ Optimum pH: 6.5-7.5 Figure 1Optimum temperature: 37℃ Figure 2pH stability: pH 6.5-9.5 (25℃,16h) Figure 3Thermal stability: Stable below 40℃ (pH8.0, 30min) Figure 4Stability: -25 ~ -15℃ standing store for 12 monthsMore than 90% activity Figure 5Protective agent: glycerin, trehalose Assay method for activity1. Principle The resulting Quinoneiminedye can be detected at 555nm.2. Definition of enzyme activityUnit enzyme activity is defined as the amount of enzyme required to catalyze the production of 1µmol H2O2 per minute under the following conditions3. Reagent preparationReagent I: 0.1M potassium phosphate buffer, pH8.0Reagent II: 1kU/mLPOD.Reagent III: 50mMTOOS solution (1.477gTOOS dissolved in 100mLUP water).Reagent IV: 50 mm4-AA solution (1.016g4-AA dissolved in 100mLUP water).Reagent V: 200mM glycosylated valine.Sample: Diluted with enzyme diluent 20mMTris-HCl, pH8.0.Prepare the reaction mixture as follows:Reagent I: 10mLReagent II: 0.1mLReagent III: 1mLReagent IV: 1mLReagent V: 10mLDouble steam water set volume to 100mLSample: Diluted with enzyme diluent 20mMTris-HCl, pH8.0.4. Operation procedure4.1 Add 980µL reaction mixture to 1mL colorimetric dish.4.2 Incubate at 37°C for 5min.4.3 Add 20µL of enzyme solution to the reaction mixture.4.4 Reaction at 4.37°C, the absorbance change (∆As) within 1min of the sample is detected by spectrophotometer at 555nm.* Replace the enzyme solution to be tested with enzyme diluent and determine the absorbance change (∆Ab) of the sample within 1min.∆A=∆As-∆Ab5. Vitality computing Vt: total volume of reaction liquid (1.0mL);Vs: enzyme liquid volume (0.02mL);t: Reaction time (1min);df: dilution ratio;C: Enzyme concentration (mg/mL);1.0: optical path length (cm);1/2:1 mole of hydrogen peroxide to generate 1/2 mole of quinone imide dye;39.2: Under standard reaction conditions, the millimolar absorption coefficient of the color group at 555nm (cm2/µmol)... Read More | Inquire | 1、Product attributeShelf life: 24 monthsReaction time：long (up to 45 minutes) at 20-37°CLot-to-lot variation：<10%Boiling point : 100℃pH-Value (at 20 °C): 9.0-9.8 Density (20℃) : 1.0302 g/cm³Water solubility: easily solubleAppearance: colourless to 1、Product attributeShelf life: 24 monthsReaction time：long (up to 45 minutes) at 20-37°CLot-to-lot variation：<10%Boiling point : 100℃pH-Value (at 20 °C): 9.0-9.8 Density (20℃) : 1.0302 g/cm³Water solubility: easily solubleAppearance: colourless to light yellow liquidOdour: odourlessIncubation temperature: 20-37 °CLight sensitiveHeat sensitive 2、Requirements for storage rooms and vessels1.Keep container tightly closed.2.Keep cool. protected from light3. Do not store together with: Oxidizing agent. 4. Contaminated or leaked out substrate solution from damaged bottles should not be used anymore and has to be destroyed.5. Use isolated containers with some cool bags for transport.6. Spontaneous decay will increase the background. If stored at room temperature, the velocity of the decay will increase. Thus, both storage and transport at room temperature should be avoided. Nevertheless, the activity of the solution is not affected by storage at room temperature. The solution still works beyond the expiry date, but some applications, especially those including visual evaluation, may be hampered by increased background. 3、Effective Components and Principle of FunctionIn different buffer solutions (pH = 9.5), with supplementation if required, the effective componentpara nitrophenyl phosphate (pNPP) is dissolved. Alkaline Phosphatase transfers the phosphate residue to an acceptor. Under alkaline conditions a yellow colour occurs, resulting from the formed nitrophenol. 4、Biosafety informationThis mixture is not classified as hazardous in accordance with Regulation (EC) No 1272/2008； 5、Advantage1. Signal yield comparable to competitor ready-to-use solutions2. Broad measurement range3. Very low background signals4. Very low blank drift during long-term storage (<0.15 AU within 24 months)5. High colour stability after reaction stop with this product and other commonly used stopping solutions 6、Instruction for usageFor bottling consider the following instructions:• Work in a dust free and darkened room.• Keep the solution as cool as possible.• Avoid contact of the solutions with any metal parts• Clean all instruments and vessels very extensively.• Wear powder-free gloves during bottling.• Close the bottles immediately to minimize the influence of light and dust.• Use clean bottles that are impermeable to light made from HDPE or PP. 7、 General Instructions for the Use in Blotting Systems Only qualified laboratory staff, who are familiar with the basics of immunological methods, are allowed to use these solutions.The substrate solutions can be used in qualitative and quantitative ELISA procedures.When using 96-well microtiter plates, adding 100 µL of substrate per well after incubation and washing is recommended. After substrate incubation the reaction can be stopped and the photometric measurement can be carried out. Using higher incubation temperatures (37° C) may shorten the incubation time. The reaction can be stopped by using the special developed solution stop. The use of other commercially available stop solutions cannot safely exclude a further increase of the signal. Addition of a stopping solution does not change the general shape of the spectrum. The unstopped and the stopped solution should be measured at 405 nm and the background correction: should be measured at 620 nm... Read More | Inquire | Purity> 95 % by SDS-PAGE and HPLC analyses.FunctionPromotes cell proliferation, chemotaxis, angiogenesis and cell adhesion. Appears to play a role in wound healing by up-regulating, in skin fibroblasts, the expression of a number of genes involved in angiogenesis, inflammation and matrix Purity> 95 % by SDS-PAGE and HPLC analyses.FunctionPromotes cell proliferation, chemotaxis, angiogenesis and cell adhesion. Appears to play a role in wound healing by up-regulating, in skin fibroblasts, the expression of a number of genes involved in angiogenesis, inflammation and matrix remodeling including VEGA-A, VEGA-C, MMP1, MMP3, TIMP1, uPA, PAI-1 and integrins alpha-3 and alpha-5. CYR61-mediated gene regulation is dependent on heparin-binding. Down-regulates the expression of alpha-1 and alpha-2 subunits of collagen type-1. Promotes cell adhesion and adhesive signaling through integrin alpha-6/beta-1, cell migration through integrin alpha-v/beta-5 and cell proliferation through integrin alpha-v/beta-3.Banckground:Cyr61, also known as CCN1, is a 40-45 kDa matricellular glycoprotein that plays an important role in cellular adhesion and migration (1). Cyr61 consists of an IGFBP domain, a VWF type C domain, a TSP type I domain, and a cysteine knot domain (2). Mature human Cyr61 shares 93% amino acid sequence identity with mouse and rat Cyr61. It is widely expressed during development and in adult tissues (2, 3). Cyr61 associates with the extracellular matrix (ECM) and with many cell surface molecules including Integrins alpha V beta 3, alpha V beta 5, alpha M beta 2, and alpha 6 beta 1, Syndecan-4, and heparan sulfate proteoglycans (1, 3). Cyr61 mediates the adhesion and migration of multiple cell types and also promotes vascular endothelial cell tubule formation (4-6). Plasmin cleavage of ECM-bound Cyr61 releases a 28 kDa N-terminal fragment which retains the ability to promote endothelial cell migration (7). Cyr61 exhibits both tumorigenic and tumor suppressor properties. It is up-regulated and promotes tumorigenesis, angiogenesis, and metastasis in breast, renal, gastric, squamous cell, and colorectal carcinomas as well as in glioma (8-12). In contrast, whendown-regulated, it suppresses tumor growth in endometrial, hepatic, and non-small cell lung cancers (8, 13, 14). Cyr61 is also up-regulated in injured skin and bone where it induces the expression of growth factors, cytokines, proteases, and integrins involved in wound repair (15, 16)... Read More |