| Description | Galactose oxidase oxidizes galactose and some galactose derivatives in both free and polymeric forms. Oxidation occurs at the C6 position. The enzyme has a molecular weight of 68 ± 3 kDa, and the optimum pH is 7.0.Useful in the determination of lactose.Application Galactose Oxidase from Galactose oxidase oxidizes galactose and some galactose derivatives in both free and polymeric forms. Oxidation occurs at the C6 position. The enzyme has a molecular weight of 68 ± 3 kDa, and the optimum pH is 7.0.Useful in the determination of lactose.Application Galactose Oxidase from Dactylium dendroides has been used as a component for galactose oxidase treatment of arabinogalactan. It has also been used to co-immobilise with peroxidase for the preparation of a biosensor for galactose detection. Galactose oxidase may be used as an analytical tool for the specific determination of D-galactose in blood plasma, plant extracts, and phospholipids. It could be used for the characterization of terminal D-galactoside units in several polymers. It may also be useful in the determination of lactose.1、Specificity ：GAO has a wide substrate specificity, but remarkable stereospecificity, only oxidizing D-isomers of substrates (McPherson et al. 1992). GAO will oxidize galactose and some galactose derivatives in both free and polymeric form. Oxidation occurs at the C6 position. 2、Composition：GAO contains one Cu(II) atom yet catalyzes a two-electron transfer reaction (McPherson et al. 1992). The copper is bound by two tyrosines, and two histidines (Tyr272, Tyr495, His496, and His581). In a novel post-translational covalent modification, Tyr272 is linked by a thioether bond to cysteine (Cys228), suggesting the involvement of a tyrosine radical in the catalytic mechanism. Stabilization of the radical occurs because Tyr272 of the thioether bond is liganded to the copper, creating a stacking interaction with Trp290 (Whittaker et al. 1989, Ito et al. 1991, and Whittaker et al. 2005). The structure of the enzyme has revealed extensive beta-sheet secondary structure, consistent with the high stability of the enzyme (Kosman et al. 1974). Most extracellular proteins of eukaryotes are modified by glycosylation during passage through the ER and golgi, leading to greater glycosylation of extracellular than intracellular forms of a protein. Unusually, the intracellular form of GAO is more highly glycosylated (9% carbohydrate) and exhibits greater stability than the extracellular form (2% carbohydrate) (Medonca and Zancan 1988). Additionally, most proteins are modified by O- and/or N-glycosylation while GAO is only modified only by O-glycosylation (Kornfield and Kornfield 1985, and McPherson et al. 1992)3、Molecular Characteristics：The gaoA gene contains a long open reading frame from +324 to +2507, including the mature protein-coding sequence (+521 to +2507). It also contains a long untranslated upstream region and a putative pro-sequence with a monobasic cleavage site (McPherson et al. 1992).4、Characteristics of Galactose Oxidase：Protein Accession Number：P0CS93；Isoelectric point：7.75 (Theoretical)CATH Classification：Three domains:Class: Mainly BetaArchitecture: Sandwich, 7 PropellorTopology: Jelly Rolls, Methylamind Dehydrogenase; Chain H, Immunoglobulin-likeMolecular Weight68.5 kDa (calculated from translated DNA sequence and SDS-polyacrylamide gel electrophoresis, McPherson et al. 1992)68.0 ± 3.0 kDa (determined from physical measurements, Cooper et al. 1959) Optimal pH：7.0 (Cooper et al. 1959) Extinction Coefficient：122,480 cm-1M-1 (Theoretical)E1%, 280 = 17.87 (Theoretical)InhibitorsCyanideDiethyldithiocarbamateAzideHydroxylamineEDTAApplicationsQuantitative determination of galactose in blood and other biological fluids (Frings and Pardue 1964, Hankin 1966, and Roth et al. 1965)Locating galactose histochemically (Roberts and Gupta 1965)Detecting and distinguishing glycoproteins (Itaya et al. 1975)5、Galactose Oxidase Assay：MethodThe reaction velocity is measured in a peroxidase/o-tolidine coupled system as an increase in A425 resulting from the oxidation of galactose. One unit results in a change in A425 of 1.0 per minute at 25°C and pH 6.0 under the defined conditions.Reagents0.1 M Potassium phosphate buffer, pH 6.00.5% o-tolidine. Note: o-tolidine has been reported to be carcinogenic. Handle with care.Peroxidase. Dissolve Worthington peroxidase (Code: HPOD) at a concentration of approximately 60 u/ml in reagent grade water.10% galactose. Allow to come to equilibrium of mutarotation by allowing to stand overnight.EnzymeDissolve at a concentration of 1 mg/ml in reagent grade water. Dilute further for assay to a concentration of 0.2 - 0.5 units/ml.ProcedureAdjust spectrophotometer to 425 nm and 25°C.Prepare tolidine-buffer mixture by adding 0.1 ml tolidine to 12 ml 0.1 M potassium phosphate buffer pH 6.0.Pipette into each cuvette as follows:Tolidine-buffer solution 1.7 ml10% Galactose 1.5 mlPeroxidase 0.1 mlIncubate in spectrophotometer at 25°C for 3 - 4 mintues to achieve temperature equilibration and establish blank rate, if any. Add 0.1 ml of appropriately diluted enzyme and record increase in A425/min. from initial linear portion of the curve... Read More | Inquire | Crude collagenase preparations contain several isoforms of two different collagenases, a sulfhydryl protease, clostripain, a trypsin-like enzyme, and an aminopeptidase. This combination of collagenolytic and proteolytic activities is effective at breaking down intercellular matrices, the essential Crude collagenase preparations contain several isoforms of two different collagenases, a sulfhydryl protease, clostripain, a trypsin-like enzyme, and an aminopeptidase. This combination of collagenolytic and proteolytic activities is effective at breaking down intercellular matrices, the essential part of tissue dissociation. One component of the complex is a hydrolytic enzyme which degrades the helical regions in native collagen preferentially at the Y-Gly bond in the sequence Pro-Y-Gly-Pro, where Y is most frequently a neutral amino acid. This cleavage yields products susceptible to further peptidase digestion. Crude collagenase is inhibited by metal chelating agents such as cysteine, EDTA or o-phenanthroline but not DFP. It is also inhibited by α2-macroglobulin, a large plasma glycoprotein. Ca2+ is required for enzyme activity. Particular enzymatic profiles of each collagenase have been correlated with the tissues from which the cells for study were obtained (or with the uses to which the cells are put) and as a result of the correlations several types of crude collagenases have been established by Aladdin: Types 1, 2, 3, and 4.This collagenase has been tested with cell lines to verify the product is not cytotoxic. Collagenase is typically used to digest the connective components in tissue samples to liberate individual cells. The concentration for cartilage dispersal is 1-2 mg/ml, but literature searches should be performed for species specific and/or tissue specific concentrations... Read More | 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 | TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for measuring HRP probe activity. A stable blue precipitate is formed at the reaction site.The substrate does not contain NMP (1-methyl2-pyrrolidone) making it REACH Restricted Substances List Annex XVII compliant, while ensuring maximal safety during use, and minimal negative environmental impact.Product Characteristics TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for measuring HRP probe activity. A stable blue precipitate is formed at the reaction site. The substrate does not contain NMP (1-methyl-2- pyrrolidone) making it REACH Restricted Substances List Annex XVII compliant, while ensuring maximal safety during use, and minimal waste problems after use.Composition & Properties Ready-to-use substrate: Includes substrate buffer and hydrogen peroxide. No other reagents should be added.Working Procedure The following procedure is applicable to nitrocellulose membranes. The procedure must be optimized for other membranes.1.The desired amount of substrate is poured into a sealed container and allowed to reach room temperature, in the dark, before use. 2.After the last incubation with HRP-labelled Streptavidin or HRP-labelled secondary antibody it is recommended to wash the membrane in a 0.1 M Tris buffer pH 7.4.3.Shake off the excess buffer and incubate the membrane in the TMB-D Blotting solution for 10 minutes. 4.Wash the membrane in distilled water and allow it to dry. 5.The site of positive reaction will appear light blue with no or very little background staining.Tips & Tricks • The membrane can be blocked with Kementec’s Synthetic Blocking Buffer for Blotting, (cat. no. S494457). • For long-term preservation of the results, the membranes must be stored in the dark.Handling & Storage • Store solution at 2-8⁰C in the dark. • Avoid exposure to light, heat and contamination with metal ions or peroxidase. • Re-dispense only into bottles made of High-Density Polyethylene (HDPE), amber color. Dispensing guidelines are available upon request... Read More |