| Description | Starch hydrolases include α-amylase (α-AL, EC 3.2.1.1) and β-amylase (β-AL). α-Amylase randomly catalyzes the hydrolysis of α-1,4-glycosidic bonds in starch, producing reducing sugars such as glucose, maltose, maltotriose, and dextrins, while simultaneously Starch hydrolases include α-amylase (α-AL, EC 3.2.1.1) and β-amylase (β-AL). α-Amylase randomly catalyzes the hydrolysis of α-1,4-glycosidic bonds in starch, producing reducing sugars such as glucose, maltose, maltotriose, and dextrins, while simultaneously reducing the viscosity of starch, hence it is also known as the liquefying enzyme. α-Amylase is widely distributed, from microorganisms to higher plants. Detection Principle: Starch hydrolases catalyze the hydrolysis of starch to produce reducing sugars. These reducing sugars reduce 3,5-dinitrosalicylic acid (DNS) to produce a brown-red-colored compound with an absorption peak at 540 nm. The amylase activity is calculated by measuring the rate of increase in absorbance at 540 nm. α-Amylase is heat-stable, but β-amylase can be inactivated by heating at 70°C for 15 minutes. Therefore, after the crude enzyme extract is treated at 70°C for 15 minutes, only α-amylase can catalyze starch hydrolysis. Detection Range: 0.0156 - 1 mg/mL Sensitivity: 0.0078 mg/mL Applicable Samples: Saliva, animal tissues, plant tissues (seeds or newly germinated seedlings) Note: The detection range and sensitivity are based on the standard. The actual detection range and sensitivity for activity need to be calculated according to the sample conditions.G1501772Component96TStorageG1501772ADNS Reagent40 mL2-8℃. Store in the dark.G1501772BSubstrate1EA2-8℃G1501772CStandard1EA2-8℃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 Reagents1.Microplate reader or visible spectrophotometer (capable of measuring absorbance at 540 nm)2.96-well plate or micro glass cuvettes, adjustable micropipettes and tips3.Centrifuge, water bath4.Deionized water5.Homogenizer (for tissue samples)Experimental Procedure1. Reagent PreparationReagent NameReagent PreparationNotesDNS ReagentReady-to-use; Equilibrate to room temperature before use.Store at 4°C protected from light.SubstrateBefore use, add 20 mL deionized water, invert and shake several times, heat until dissolved.Unused reagent can be stored at 4°C for one week. If precipitate forms, heat to 70°C to dissolve.StandardBefore use, add 1 mL deionized water to dissolve, obtaining a 10 mg/mL standard (Glucose) stock.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 1, 0.5, 0.25, 0.125, 0.0625, 0.0313, and 0.0156 mg/mL as shown in the table below.TubeStandard VolumeDeionized Water Volume (µL)Standard Concentration (mg/mL)Std.140µL (10 mg/mL)3601Std.2200µL of Std.12000.5Std.3200µL of Std.22000.25Std.4200µL of Std.32000.125Std.5200µL of Std.42000.0625Std.6200µL of Std.52000.0313Std.7200µL of Std.62000.0156Note: The standard curve must be generated with each experiment. Diluted standard solutions are unstable and must be used within 4 hours3. Sample PreparationNote: Fresh samples are recommended.3.1 Animal TissueWeigh approximately 0.1 g of tissue. Add 1 mL of deionized water and homogenize. Transfer the homogenate to a centrifuge tube. Let it stand at room temperature for 15 minutes, vortexing every 5 minutes for sufficient extraction. Centrifuge at 6,000 g for 10 minutes at room temperature. Aspirate the supernatant and dilute to 10 mL with deionized water. Mix well. This is the amylase stock solution.3.2 Plant TissueWeigh approximately 0.1 g of tissue. Add 1 mL of deionized water and grind. Sonicate for 5 minutes (power 20%, pulse 3s on, 7s off, repeat 30 times). Let it stand at room temperature for 15 minutes, vortexing every 5 minutes for sufficient extraction. Centrifuge at 6,000 g for 10 minutes at room temperature. Aspirate the supernatant and dilute to 10 mL with deionized water. Mix well. This is the amylase stock solution.3.3 Saliva, and Other Liquid SamplesAssay directly. It is recommended to perform a preliminary test to determine the appropriate dilution factor.Note:For animal tissues with high fat content, remove the upper fat layer after centrifugation before collecting the supernatant.If protein concentration measurement is required, use Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648).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 Preheat a water bath to 70°C.4.3 Take 75 µL of sample and incubate in a boiling water bath for 5 minutes. This will be used as the Control tube.4.4 Sample Measurement (Add reagents sequentially into microcentrifuge tubes as below):ReagentBlank Tube (µL)Standard Tube (µL)Test Tube (µL)Control Tube (µL)Deionized Water75000Standard (various conc.)07500Sample007575 (boiled sample)Heat at 70°C for 15 min, then cool.Substrate00750Incubate in a constant temperature water bath at 40°C for 5 min.DNS Reagent150150150150Substrate75750754.5 Mix well. Incubate in a boiling water bath for 5 minutes. Cool. 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: Each sample requires a control tube. The blank tube only needs to be prepared once. It is recommended to perform a preliminary test with 2-3 samples expected to have significant differences before the formal experiment. If A test > 2, the enzyme activity is too high, and the sample must be diluted with deionized water to an appropriate concentration (multiply by the dilution factor in the calculation). If ΔA test < 0.005, re-extract the sample reducing the final volume of deionized water used for dilution.5. Calculation of Results 5.1 Standard Curve Plotting Plot 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 α-Amylase Activity Calculation (1) Based on Sample Fresh Weight Calculation (1) Based on Sample Fresh Weight Calculation Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 mg of reducing sugar per minute per gram of tissue. Calculation Formula: α-Amylase Activity (U/g weight) = y × V sample ÷ (W × V sample ÷ V total ) ÷ T × n = 2 × y ÷ W × n (2) Based on Sample Protein Concentration (2) Based on Sample Protein Concentration Calculation Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that produces 1 mg of reducing sugar per minute per milligram of tissue protein. Calculation Formula: α-Amylase Activity (U/mg prot) = y × V sample ÷ (Cpr × V sample ) ÷ T × n = 0.2 × y ÷ Cpr × n (3) Based on Liquid Sample Volume Calculation Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that produces 1 mg of reducing sugar per minute per liter of liquid sample. Calculation Formula: α-Amylase Activity (U/L) = 1000 × y ÷ T × n = 200 × y × n Parameter Definitions: y: Concentration of reducing sugar calculated from the standard curve (mg/mL) V sample : Volume of sample added to the reaction system (0.075 mL) W: Sample weight (g) V total : Total volume of the sample extract (10 mL) T: Enzymatic reaction time (5 minutes) n: Sample dilution factor Cpr: Sample protein concentration (mg/mL) 1000: Conversion factor between liters and milliliters (1 L = 1000 mL)6. Representative ResultsTypical Standard Curve: y = 0.4948x - 0.0179, R² = 0.9982Precautions1. Biochemical reagents are generally irritating and potentially biologically toxic. For your safety and health, please use appropriate biosafety precautions throughout the experiment, including wearing lab coats, masks, gloves, and head covers. Perform experiments in a fume hood or biosafety cabinet.2. This product is for research use only. Not for use in clinical diagnosis... Read More | Product introduction：Product introduction：Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) has certain applicability for live cells and fixed cell cycle detection. For different types of cells, whether it is applicable or not needs to be determined after testing. Cell Cycle Product introduction：Product introduction：Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) has certain applicability for live cells and fixed cell cycle detection. For different types of cells, whether it is applicable or not needs to be determined after testing. Cell Cycle Assay Kit Plus ( Cell Cycle Assay Kit Plus ) uses RedNucleus I staining to detect cell cycle. RedNucleus I is a far-infrared nucleic acid dye with cell membrane permeability, which can quickly enter living cells, specifically bind to DNA, and perform cell cycle detection on living cells without RNase digestion. Compared with the traditional PI staining method, the cells do not need to be broken or fixed, and the operation is simpler. RedNucleus I is a fluorescent dye of double-stranded DNA, and the fluorescence intensity after binding to double-stranded DNA is proportional to the content of double-stranded DNA. The intracellular DNA content can be measured by flow cytometry, and then the cell cycle analysis can be carried out according to the distribution of DNA content. After RedNucleus I staining, assuming that the fluorescence intensity of G0 / G1 phase cells is 1, the theoretical value of the fluorescence intensity of G2 / M phase cells containing two copies of genomic DNA is 2, and the fluorescence intensity of S phase cells undergoing DNA replication is between 1-2. In addition, RedNucleus I is compatible with dyes such as Horizon BV / BUV, FITC and R-PE, and can be periodically detected after sample staining.The kit is usually used to detect the cell cycle of cultured adherent or suspended cells. If it is used for cell cycle detection of tissues, the tissues must be digested into a single cell state.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. this product is applicable to the detection of living cells and fixed cell cycle with certain limitations. Whether it is applicable to different types of cells needs to be determined after testing. If fixation is needed, it is recommended to use ice bath pre cooling 75-80% ethanol -20 ℃ to fix cells overnight. 3. fluorescent dyes have quenching problems. Please try to avoid light during storage and use to slow down fluorescence quenching. 4. for your safety and health, please wear experimental clothes and disposable gloves.Instruction： Experimental materials ( self-provided )：①cell lines or other cell samples ( self-prepared ) ；②This kit ; ③ trypsin ( self-prepared ) ;④ Cell culture medium containing FBS ( self-prepared ) ; Experimental procedure： 1.Preparation of cell samples : ( 1 ) ( This step is for adherent cells, if suspended cells, can be carried out directly step ( 2 ) ) Digest cells with trypsin, add cell culture medium, gently blow away cells, collected into the centrifuge tube. Note : The number of cells on the machine needs to reach 50,000 and above, so the initial number of cells collected needs to be sufficient. ( 2 ) Centrifuged about 1000 g for 3-5 min to precipitate cells. Carefully remove the supernatant, add about 1 mL of ice bath pre-cooled 1 × staining buffer ( 10 × staining buffer diluted with diH2O at 1 : 10 ), re-suspend the cells. Repeat once. ( 3 ) Centrifuged about 1000 g for 3-5 min to precipitate cells. After the supernatant was discarded, 1 mL of culture medium was added to re-suspend the cells ( for fixed cells, 1 × PBS can also be used to re-suspend ). Gently flick the bottom of the centrifuge tube to properly disperse the cells to avoid cell aggregation. 2.Staining : 4 µL of RedNucleus I staining solution was added to each tube of cell samples, slowly and fully mixed, and incubated at room temperature in dark for 20 min ( or incubated at 37 ° C in dark for 5-10 min ). The optimal incubation time of different cells is different, and the staining time can be adjusted and optimized according to the actual staining effect to obtain a more ideal staining effect. 3.Flow cytometry detection and analysis : Excited at 638 nm by flow cytometry, it is recommended to detect in RL3 or FL4 channels, or use RL1 and RL2 channels. Cell DNA content analysis and light scattering analysis were performed using appropriate analysis software.Scope of application：Cell cycle detection... Read More | Inquire | This reagent kit is for research purposes only. Purpose of use: This reagent kit is used to determine the content of lactose in serum, plasma, and related liquid samples.Experimental principle:This kit applies enzyme-linked immunosorbent assay to determine the level of lactose in the sample. This reagent kit is for research purposes only. Purpose of use: This reagent kit is used to determine the content of lactose in serum, plasma, and related liquid samples.Experimental principle:This kit applies enzyme-linked immunosorbent assay to determine the level of lactose in the sample. Purified lactose antibodies were coated on a microplate to produce solid-phase antibodies. Lactose was added to the microplate of the coated monoclonal antibody, along with HRP labeled lactose antigens, to compete for binding. After thorough washing, the substrate TMB was added for colorimetry. The depth of sample color is negatively correlated with the content of lactose in the sample. Measure the absorbance (OD value) at a wavelength of 450nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the content of lactose in the sample through a standard curve.Kit composition:130times concentrated washing solution20ml×1 bottle8.1Standard S1（80µg/L）0.5ml×1bottle2Enzyme-linked immunosorbent assay6ml×1 bottle8.2Standard S2（40µg/L）0.5ml×1bottle3Enzyme labeling coated plate96 holes x 1 pieces8.3Standard S3（20µg/L）0.5ml×1bottle4Color reagent A solution6ml×1 bottle8.4Standard S4（10µg/L）0.5ml×1bottle5Color developer B solution6ml×1 bottle8.5Standard S5（5µg/L）0.5ml×1bottle6Stop solution6ml×1 bottle9Instructions1 copy7Sample Diluent6ml×1 bottle10Microplate Sealers2 sheetsSpecimen requirements:1. Specimen processing:(1) After collecting the water sample, it is repeatedly freeze-thawed three times at -20 ℃, and then filtered through glass fiber for future reference(2) The tissue samples should be extracted using butanol: methanol: water (5:25:70 V: V: V), or extracted according to relevant literature. The experiment should be conducted as soon as possible after extraction. If the experiment cannot be conducted immediately, the specimen can be stored at -20 ℃ for future reference2. Samples containing NaN3 cannot be detected as NaN3 inhibits the activity of horseradish peroxidase (HRP).Operation steps:1. Sample addition: Set up standard wells, blank wells (blank control wells do not include samples and enzyme-linked immunosorbent assay reagents, the other steps are the same), and sample wells to be tested. Add 50 microliters to the standard well on the enzyme-linked immunosorbent assay (ELISA) plate, and first add 40 diluents to the sample well to be tested µ l. Then add 10 more samples to be tested µ L (The final dilution of the sample is 5 times). Add the sample to the bottom of the enzyme-linked immunosorbent assay (ELISA) plate well, avoiding touching the well wall as much as possible. Gently shake and mix well.2. Enzyme addition: Add 50 enzyme labeled reagents to each well µ l. Excluding blank holes.3. Warm incubation: Seal the plate with a sealing film and incubate at 37 ℃ for 60 minutes.4. Solution preparation: Dilute 30 times the concentrated washing solution with distilled water and set aside for later use5. Washing: Carefully remove the sealing film, discard the liquid, shake dry, fill each well with washing solution, let it stand for 30 seconds, then discard. Repeat this process 5 times and pat dry.6. Color development: Add color development agent A50 to each well first µ l. Add color developer B50 again µ l. Gently shake and mix well, and develop color at 37 ℃ in the dark for 15 minutes7. Termination: Add 50% termination fluid to each hole µ l. Terminate the reaction (at this point, the blue color immediately turns yellow).8. Measurement: Zero the blank hole and sequentially measure the absorbance (OD value) of each hole at a wavelength of 450nm. The measurement should be conducted within 15 minutes after adding the termination solution.Calculation:Draw a standard curve on a coordinate paper with the concentration of the standard substance as the x-axis and the OD value as the y-axis. Based on the OD value of the sample, determine the corresponding concentration from the standard curve; Multiply it by the dilution factor; Alternatively, a linear regression equation can be used to calculate the standard curve using the concentration and OD value of the standard substance. The OD value of the sample can be substituted into the equation to calculate the sample concentration, which is then multiplied by the dilution factor to obtain the actual concentration of the sample.Notes:1. The kit should be balanced at room temperature for 15-30 minutes before use when taken out from the cold storage environment. If the enzyme coated plate is not used up after opening, the Flat noodles should be stored in a sealed bag.2. Concentrated detergent may precipitate crystals. When diluted, it can be heated in a water bath to aid in dissolution. Washing does not affect the results.3. A sampler should be used for each step of sample addition, and its accuracy should be regularly calibrated to avoid experimental errors. It is best to control the sample addition time within 5 minutes. If there are a large number of specimens, it is recommended to use a firing gun for sample addition.4. Please make a standard curve at the same time as each measurement, preferably with a re hole. If the content of the substance to be tested in the sample is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please dilute the sample diluent by a certain multiple (n times) before measurement. When calculating, please multiply the total dilution multiple (x n x 5).5. The sealing film is only for one-time use to avoid cross contamination.6. Please store the substrate in dark.7. Strictly follow the instructions and determine the test results based on the reading of the enzyme-linked immunosorbent assay (ELISA) reader8. All samples, washing liquids, and various waste should be treated as infectious substances.9. The components of this reagent with different batch numbers shall not be mixed.Detection range:two µ G/L-90 µ G/L... Read More | Inquire |