| Description | Reducing sugars (RS) are widely present in animals, plants, microorganisms, and cultured cells. Reducing sugars in plants primarily include glucose, fructose, and maltose. Among these, glucose and fructose are not only the main substrates for respiration but also serve as substrates for the further Reducing sugars (RS) are widely present in animals, plants, microorganisms, and cultured cells. Reducing sugars in plants primarily include glucose, fructose, and maltose. Among these, glucose and fructose are not only the main substrates for respiration but also serve as substrates for the further synthesis of sucrose, starch, and cellulose.Detection Principle: In an alkaline solution, 3,5-dinitrosalicylic acid (DNS) can be reduced by reducing sugars to produce a brown-red-colored amino compound, which has a characteristic absorption peak at 540 nm. Within a certain concentration range, the RS content is linearly correlated with the absorbance at 540 nm. The RS content in the sample can be calculated based on a standard curve.Detection Range: 0.05 - 0.6 mg/mLSensitivity: 0.025 mg/mLApplicable Samples: Plant tissues, animal tissues, cells, bacteria, serum (plasma)R1501790Component48T96TStorageR1501790AExtraction Buffer60 mL120 mL2-8℃R1501790BDNS Reagent10 mL20 mL2-8℃. Store in the dark.R1501790CStandard1EA1EA2-8℃Note: Before formal testing, it is recommended to perform a preliminary test with 2-3 samples expected to have significant differences.User-Prepared Instruments and ReagentsMicroplate reader or visible spectrophotometer (capable of measuring absorbance at 540 nm)96-well plate or micro glass cuvettes, adjustable micropipettes and tipsCentrifuge, water bathDeionized waterHomogenizer (for tissue samples)Experimental Procedure1. Reagent PreparationReagent NameReagent PreparationNotesExtraction BufferReady-to-use; Equilibrate to room temperature before use.Store at 4°C. Slightly irritating. Use appropriate personal protective equipment.DNS ReagentReady-to-use; Equilibrate to room temperature before use.Store at 4°C protected from light. Slightly irritating. Use appropriate personal protective equipment.StandardBefore use, add 1 mL of deionized water to dissolve, preparing a 10 mg/mL stock standard solution.Can be stored at 4°C for 2 weeks.2. Standard Curve SetupDilute the 10 mg/mL standard stock solution with deionized water to concentrations of 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, and 0.05 mg/mL.TubeVolume of 10 mg/mL Standard (µL)Volume of Deionized Water (µL)Concentration (mg/mL)Std.1609400.6Std.2509500.5Std.3409600.4Std.4309700.3Std.5209800.2Std.6109900.1Std.759950.05Note: The standard curve must be generated with each experiment. Diluted standard solutions are unstable and must be used within 4 hours.3. Sample Preparation3.1 Plant or Animal Tissue SamplesWeigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and homogenize in an ice bath. Transfer the homogenate to a capped centrifuge tube (to prevent evaporation during heating). Incubate in an 80°C water bath for 40 minutes, vortexing every 5 minutes. Centrifuge at 8,000 g, 25°C for 10 minutes. Collect the supernatant for assay.3.2 Bacteria or CellsCollect bacteria or cells into a centrifuge tube; discard the supernatant. Add 1 mL of Extraction Buffer per 5 million bacteria/cells. Sonicate in an ice bath for 5 minutes (power 20%, pulse 3s on, 10s off, repeat 30 times). Transfer to a capped centrifuge tube (to prevent evaporation during heating). Incubate in an 80°C water bath for 40 minutes, vortexing every 5 minutes. Centrifuge at 8,000 g, 25°C for 10 minutes. Collect the supernatant for assay.3.3 Serum (Plasma) SamplesTake 0.1 mL of serum (plasma) and add 0.9 mL of Extraction Buffer; mix thoroughly. Transfer to a capped centrifuge tube (to prevent evaporation during heating). Incubate in an 80°C water bath for 40 minutes, vortexing every 5 minutes. Centrifuge at 8,000 g, 25°C for 10 minutes. Collect the supernatant for assay.Note:If protein concentration measurement is required, Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended. The Extraction Buffer contains components that denature proteins. If calculating based on protein concentration, protein needs to be re-extracted separately for measurement.4. Assay Steps4.1 Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 540 nm. For spectrophotometers, zero the instrument with deionized water.4.2 Assay Procedure:ReagentBlank Tube (µL)Standard Tube (µL)Test Tube (µL)Control Tube (µL)Sample00175175Standard (various conc.)017500Deionized Water17500125DNS Reagent1251251250Mix well. Heat in a boiling water bath for 5 minutes (cap tightly to prevent evaporation). Remove and immediately cool to room temperature. Transfer 200 µL to a 96-well plate or micro glass cuvette. Measure the absorbance at 540 nm. Calculate ΔA test = A test - A control, ΔA standard = A standard - A blank. Note:The Blank and Standard tubes only need to be set up 1-2 times.It is recommended to perform a preliminary test with 2-3 samples expected to have significant differences before the formal experiment. If ΔA <sub> test </sub> is less than 0.04, consider increasing the sample volume appropriately. If ΔA <sub> test </sub> is greater than the ΔA <sub> standard </sub> of the 0.6 mg/mL standard, further dilute the sample with Extraction Buffer (multiply the result by the dilution factor) or reduce the amount of sample used for extraction.5. Calculation of ResultsNote: We provide both the derived formula and a simplified formula. They are equivalent. It is recommended to use the simplified formula in bold for final calculation.5.1 Standard Curve PlottingPlot the standard concentration (y-axis) against ΔA standard (x-axis) to generate the standard curve. Substitute ΔA test into the standard curve equation to calculate y (mg/mL).5.2 Sample Reducing Sugar Content Calculation(1) Based on Sample WeightReducing Sugar (µg/g) = 1000 × y × V<sub>extraction</sub> ÷ W × n = 1000 × y / W × n(2) Based on Sample Protein ConcentrationReducing Sugar (µg/mg prot) =1000 × y × Vextraction ÷ (Vextraction × Cpr) × n=1000 × y / Cpr × n(3) Based on Bacterial or Cell CountReducing Sugar (µg/10⁴) =1000 × y × V<sub>extraction</sub> ÷ 500 × n = 2 × y × n(4) Based on Serum (Plasma) VolumeReducing Sugar (µg/mL) = 1000 × y × Vextraction ÷ Vliquid × n = 10000 × y × nParameter Definitions:1000: Unit conversion factor (1 mg/mL = 1000 µg/mL)V extraction : Volume of Extraction Buffer added (1 mL)V liquid : Volume of serum (plasma) added (0.1 mL)Cpr: Sample protein concentration (mg/mL)W: Sample weight (g)500: Total number of bacteria or cells (5 million)n: Dilution factor6. Representative ResultsTypical Standard Curve: y = 0.2243x + 0.0545, R² = 0.9957 PrecautionsThis product is for research use only. Not for use in clinical diagnosis. For your safety and health, please wear lab coats and disposable gloves during operation... Read More | Product Descriptionalpha-L-fucoside fucohydrolase, alpha-L-fucosidase, alpha-(1-3,4) fucosidaseAlpha (1-3,4) Fucosidase The enzyme is very efficient and recognises α1-3,4 fucosylated glycans (e.g. Lewis X/A epitopes, including their sialylated counterparts) and hydrolyses terminal α1-3 andProduct Descriptionalpha-L-fucoside fucohydrolase, alpha-L-fucosidase, alpha-(1-3,4) fucosidaseAlpha (1-3,4) Fucosidase The enzyme is very efficient and recognises α1-3,4 fucosylated glycans (e.g. Lewis X/A epitopes, including their sialylated counterparts) and hydrolyses terminal α1-3 and α1-4 fucosyl linkages in these substrates without the need to remove sialic acid moieties.For removing core fucose linked α-(1-6) to the core GlcNAc of a GlcNAc-GlcNAc disaccharide structure we recommend our Alpha-(1-6) Fucosidase.• Non-sialidase dependant hydrolysis of antennary fucose moieties• Effective on both glycopeptides and free glycans• Highly specific (α1-3,4 fucosylated glycans)• Kit includes enzyme plus reaction buffer.• Sufficient for up to 50 samplesα(1-3,4) Fucosidase is useful for:nbsp;nbsp;Fucose linkage determinationnbsp;nbsp;Deglycosylating glycoproteins with Lewis structuresContentsAlpha-(1-3,4)-Fucosidase – 200 mM citrate buffer pH 6 containing 250 mM NaCl5x Reaction Buffer – 250 mM sodium phosphate pH 6... Read More | The Endo F Multi-Kit will deglycosylate N-linked glycans in both native and denatured conditions. Each enzyme has a distinct specificity for N-linked glycan release. One can choose to use the three enzymes in combination to completely remove all N-linked glycans present on a glycoprotein or peptide,The Endo F Multi-Kit will deglycosylate N-linked glycans in both native and denatured conditions. Each enzyme has a distinct specificity for N-linked glycan release. One can choose to use the three enzymes in combination to completely remove all N-linked glycans present on a glycoprotein or peptide, or to use each enzyme independently and thereby determine the type of N-glycans present.Product DescriptionThe Endo F Multi-kit is recommended to deglycosylate native proteins that are resistant to PNGase F cleavage under non-denatured conditions due to the glycan location within the protein’s three-dimensional structure, as these enzymes are known to be less sensitive to protein conformation.Each of the enzymes has a different N-linked glycan specificity:Endoglycosidase F1 cleaves high mannose and some hybrid type N-glycansEndoglycosidase F2 releases biantennary and high mannose glycans (at a 40X reduced rate)Endoglycosidase F3 will release triantennarry and fucosylated biantennary N-glycansContents1 vial: Endo F1- 20 µl (0.3 U)20 mM Tris-HCl pH 7.51 vial: Endo F2- 20 µl (0.1 U)10 mM sodium acetate, 25 mM NaCl, pH 4.51 vial: Endo F3- 20 µl (0.1 U)20 mM Tris-HCl pH 7.51 vial: 5x Reaction Buffer - 400 µl250 mM sodium acetate, pH4.51 vial: 5x Reaction Buffer - 400 µl250 mM sodium phosphate, pH5.5Specific ActivityDefined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micro-mole of denatured Ribonuclease B (Endo F1) or porcine fibrinogen peptides (Endo F2/F3) in 1 minute at 37°C, pH 5.5 (PH 4.5 for Endo F3). Cleavage is monitored by SDS-PAGE.FormulationThe enzymes are provided as a sterile-filtered solution.StabilitySeveral days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly.SpecificityEndo F1 cleaves Asparagine-linked (N-linked) high mannose or hybrid oligosaccharides. Endo F2 cleaves N-linked biantennary oligosaccharides and high mannose (at a 40X reduced rate). Endo F3 cleaves free or N-linked fucosylated biantennary or triantennary oligosaccharides,as well as triamannosylchitobiose core structures. These enzymes cleave between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. The recombinant version is not glycosylated, which may result in properties differing from the native protein.Quality & PurityEndo F1, Endo F2, and Endo F3 are tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37°C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The absence of exoglycosidase contaminants is confirmed by extended incubations with the corresponding pNP-glycosides. Directions for use 1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 34 µl final volume with de-ionized water. 2. Add 10 µl Endo F2 &F3 5x Reaction Buffer, 250 mM sodium acetate pH 4.5. Use Endo F1 buffer, 250 mM sodium phosphate pH 5.5 if you are using the Endo F1 enzyme alone. 4. Add 2.0 µl of each enzyme to the reaction. Incubate 3 hours at 37°C. Monitor cleavage by SDS-PAGE. Applications– Deglycosylation of native proteins resistant to PNGase F cleavage– Determination of glycan type (high mannose, biantennary, tri/tetrantennary)– Deglycosylating proteins which normally precipitate when deglycosylating– X-Ray CrystallographyThese three enzymes cleave asparagine-linked (N-linked) oligosaccharides between the two GlcNAc residues in the core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine, enhancing the solubility of the protein. In contrast, PNGase F removes the oligosaccharide intact... Read More | This product can rapidly, gently, and efficiently lyse mammalian cells, effectively extracting cytoplasmic and nuclear proteins. This reagent uses a mild formula to ensure that the extracted protein maintains biological activity and can be applied to various protein analysis experiments, such as This product can rapidly, gently, and efficiently lyse mammalian cells, effectively extracting cytoplasmic and nuclear proteins. This reagent uses a mild formula to ensure that the extracted protein maintains biological activity and can be applied to various protein analysis experiments, such as reporter gene and enzyme activity determination, immune detection, protein purification, etc. The extracted protein can be quantitatively analyzed using the BCA method. The reagent kit contains a mixture of protease inhibitors, which can effectively prevent protein degradation during the protein extraction process.M665813Component100 TStorageM665813AMammalian Protein Extraction Reagent100 mLRTM665813BProtease Inhibitor Cocktail (100×)1 mL-20℃. Avoid freeze/thaw cycle. precautions1. This product can effectively lyse adherent cells cultured on cell culture plates (without scraping) and suspended cells collected by centrifugation, with higher extraction efficiency than repeated freeze-thaw or ultrasound methods. But for the extraction of tissue proteins, it is recommended to use the tissue protein extraction kit (CW0891).The optimal dosage for protein extraction from adherent cells is listed in Table 1. Collecting cells first can reduce the amount of reagents used to obtain higher protein concentrations.3. The amount of extraction reagents used can also be estimated based on the number of cells. If 2 × 106 Hela cells weigh about 20 mg, 200 need to be added µ Extract reagents.4. The protein extracted from this product can be quantitatively analyzed using the BCA method.Operation steps● Protein extraction from adherent cells1. Please remove the required Mammalian Protein Extraction Agent for pre cooling before protein extraction.2. Carefully pour out the culture medium of adherent cells and rinse the cells with PBS.3. Add an appropriate amount of Mammalian Protein Extraction Reagent (add Protein Inhibitor Cocktail in a 1:99 ratio 2-3 minutes before protein extraction), blow adherent cells on ice with a gun tip, transfer the lysate to a centrifuge tube, incubate on ice for 20 minutes, and allow the cells to fully lyse (please refer to Appendix 1 for the amount of reagent used, and the time for placing on ice should be adjusted according to different cell types). 4. Centrifuge at 14000 × g for 5-10 minutes.5. Transfer the supernatant to a new tube for further analysis. ● Suspension cell protein extraction1. Please remove the required Mammalian Protein Extraction Agent for pre cooling before protein extraction.2. Suspend 2500 × g of cells, centrifuge for 10 minutes, and discard the supernatant. Rinse cells with PBS. 2500 × g, centrifuge for 10 minutes, discard the supernatant.3. Add an appropriate amount of Mammalian Protein Extraction Agent, and 2-3 minutes before protein extraction, add Protein Inhibitor Cocktail in a ratio of 1:99, which is 1 x working solution.4. Add at least 1 ml of 1x working solution to every 100 mg of cells. If the extracted sample size is large, a small amount of 1x working solution can be used to resuspend the cells first, and then the remaining working solution can be added.5. After blowing evenly, place it on ice for 20 minutes to allow the cells to fully lyse (the time for placing it on ice should be adjusted according to different cell types). 6. Centrifuge at 14000 × g for 15 minutes.7. Transfer the supernatant to a new tube for further analysis.Table 1. Recommended usage of extraction reagents Cell culture plate type or dish type Extraction reagent usage 100 mm 500-1,000 µl 60 mm 250-500 µl 6-well culture plate 200-400 µl /well 24-well culture plate 100-200 µl /well 96-well culture plate 50-100 µl /well Table 2. Common Problems and Solutions Problem Possible reasons Resolvent Low extraction rate Low protein expression level Optimize transfection system Low extraction rate Insufficient reagent usage Increase the usage of extraction reagents Low extraction rate Reagent unable to dissolve cell membrane Increase cracking time or increase shaking amplitude Unable to obtain membrane protein This product is more suitable for extracting nuclear plasma protein Using eukaryotic cell membrane protein extraction kit... Read More | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export |