| 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 | Inquire | ProductsThis product is a high purity genomic DNA extract from 293T cells, agarose gel (0.7%) electrophoresis showed that the size of the DNA extract is more than 15Kb, and basically no degradation, the product is ultimately preserved in TE Buffer, which can be widely used in molecular biology ProductsThis product is a high purity genomic DNA extract from 293T cells, agarose gel (0.7%) electrophoresis showed that the size of the DNA extract is more than 15Kb, and basically no degradation, the product is ultimately preserved in TE Buffer, which can be widely used in molecular biology experiments, such as PCR, enzyme digestion, hybridization, microarray analysis, and other molecular biology experiments.The product was quantified using NanoDrop One at a concentration of 200 ng/µL.Preparation and precautions before useLong-term storage at -20˚C is recommended. Before use, the bottle should be removed from the refrigerator and equilibrated to room temperature and centrifuged before opening the cap for use. Samples should be restored to the sealed state as soon as possible after opening.How to use (take qPCR experiment as an example)1. Amplification template preparationThe samples to be detected were diluted with TE (10 mM Tris-Cl, pH 8.0,1 mM EDTA), and the concentration after dilution was as close as possible to the range of 0.05-10 ng/µL. The samples were placed on ice at 4°C and set aside.2. Standard dilution: according to the following table, firstly dilute Human DNA Standard 1 (100ng/uL) with TE to make 5 different concentrations of standards according to the table below. 10ng/µL of DNA Standard 1 (Std. 1) can be stored stably at -20℃ for 1 month; Std2-5 can only be used on the same day, and should be placed at 4℃ or on ice when not in use for the time being after preparation. When not used temporarily after preparation, it should be stored at 4℃ or on ice.styleCorresponding concentration (ng/µL)Minimum dilution volume (in µL)Std.11010 [100 ng/µL DNA Standard 1] + 90 TEStd.22.520 [Std. 1] +60 TEStd.30.62520 [Std. 2] +60 TEStd.40.1562520 [Std. 3] +60 TEStd.50.039062520 [Std. 4] +60 TE3. qPCR reaction system preparationThe cryopreserved reagents to be used were completely thawed and mixed by inversion several times before preparation, and then briefly centrifuged and prepared for use. 20 µL of the base reaction system was as follows.The base reaction system for 20 µL was as follows:reagents20µL reaction system2×qPCRMix10µLPrimerMixXµLProbeMixXµLTemplate4µLddH2OMake up to 20 µLNote: High Rox model: add 1 µL of 50×High Rox per 50 µL of reaction system; Low Rox model: add 1 µL of 50×High Rox per 500 µL of reaction system.Usually, better results can be obtained with a primer concentration of 0.2 µM, and 0.1-1.0 µM can be used as a reference for setting the range.The concentration of the probe used is related to the fluorescent quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, so please refer to the manual of the instrument or the specific requirements for the use of each fluorescent probe for the adjustment of the concentration during actual use.Prepare a sufficient amount of reaction system mixture as required. After the reaction system has been prepared and mixed thoroughly, add 16 µL per well to the reaction wells. Then add the prepared standard and diluted sample into the corresponding reaction wells, the volume of addition is 4µL/well. TE was added to the blank control tube, and the same amount of TE was added at 4 µL/well.It is recommended to use 20 µL for the reaction, if you need to perform a smaller system reaction, reduce the system components in equal proportion.4. qPCR reaction programThe following is an example of our GoldStar Probe Mixture reaction conditions, which should be improved and optimized according to the PCR product template, primer structure and target fragment size.movetemptimingcirculatepremutability95°C10min1denaturation95°C10sec55Annealing/Extension60°C30sec5Data analysis1. Standard curve productionThe standard curve was plotted with reference to the Excel sheet for data processing. The correlation coefficient R2 of the standard curve should not be lower than 0.98, and the slope should be between -3.1 and -3.6 when the Ct value is the vertical coordinate. If the parameters of the standard curve are unreasonable, it is recommended to repeat the experiment... Read More | Inquire |