| 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 | Inquire | Bacterial protein extraction reagents use mild non-ionic detergents and are suitable for extracting recombinant proteins expressed in Escherichia coli and insect cells. During the extraction process, there is no need for ultrasonic fragmentation, effectively avoiding contamination of exogenous Bacterial protein extraction reagents use mild non-ionic detergents and are suitable for extracting recombinant proteins expressed in Escherichia coli and insect cells. During the extraction process, there is no need for ultrasonic fragmentation, effectively avoiding contamination of exogenous proteins. This product can be applied to extract soluble proteins from bacterial lysates. The bacterial protein extraction kit adds a mixture of lysozyme, DNase I, and protease inhibitors to the extraction reagent, which can improve the efficiency of protein extraction and reduce the viscosity caused by DNA, effectively avoiding protein degradation. The extracted protein maintains biological activity and can be subjected to downstream operations such as IP, Western blot, and protein purification. Component B665764 100 preps Bacterial Protein Extraction Reagent 100 ml Protease Inhibitor Cocktail (100x) 1 ml Lysozyme （50 mg/ml） 200µl DNaseⅠ（1,000 U/ml） 100µl Notes:1. This product is suitable for extracting proteins from fresh or frozen bacterial and insect cells.2. This product uses Tris buffer system. Please use the same buffer system for protein purification after extraction.3. The protein lysis solution obtained from this product can be used for protein quantification using BCA or Bradford method.4. For special strains, if the extraction effect is not ideal, the sample can be frozen before protein extraction.5. Depending on the specific situation, protease inhibitors, salts, chelating agents, reducing agents, etc. can be added to this product.Operation steps: ● Insect cell protein extraction1. Collect cells by low-speed centrifugation. Add 10 to every 1 ml of Bacterial Protein Extraction Agent µ The Protein Inhibitor Cocktail is 1 x working fluid.2. Weigh the wet weight of the cells and add 1 x working solution at a rate of 10 ml/g.3. After resuspension, incubate on ice for 20 minutes (the ice storage time should be adjusted according to different cell types).Centrifuge at 4.15000 × g for 15 minutes to isolate soluble proteins. ● Extraction of soluble bacterial proteins 1. Centrifuge for 10 minutes at a rate of 5000 × g and collect the bacterial cells.2. Optional steps: Add 1 ml of Bacterial Protein Extraction Reagent every 1 ml µ DNase I (1000 U/ml), 2 µ Lysozyme (50 mg/ml) and 10 µ Protein Inhibitor Cocktail, vortex oscillation and mixing. 3. Add 20 ml of Bacterial Protein Extraction Reagent to each gram of bacterial precipitate, and add the extraction solution to the bacterial precipitate. Vortex thoroughly or use a pipette to blow up and down until the bacterial precipitate is completely resuspended.4. After resuspension, incubate at room temperature for 10-15 minutes (the storage time should be adjusted according to different cell types). 5. Centrifuge at 15000 × g for 5 minutes.6. Transfer the supernatant to a new centrifuge tube (the supernatant is soluble protein) for protein quantification and downstream experiments.Note: If the target protein exists in the form of inclusion bodies, inclusion body protein solution can be used for dissolution or expression conditions can be optimized to increase the expression of soluble proteins.Frequently asked questions: Problem Possible reasons Resolvent The target protein is insoluble The target protein is expressed as an inclusion body Optimize expression conditions or add Lysozyme and DNase I to protein extraction reagents using inclusion body protein solution After adding Lysozyme, the target protein has not been extracted yet Temperature too low Restore the reagent to room temperature After adding Lysozyme, the target protein has not been extracted yet Lysozyme Decreased or inactivated activity Add more Lysozymes or replace with new enzymes Extract has high viscosity DNase I Decreased or inactivated activity Add more DNase I or replace with a new DNase I to increase the final concentration of magnesium ions to 2 mM After protein extraction, most of the proteins still exist in the precipitate Excessive protein content Add Lysozyme and DNase I The protein extraction reagent has sediment precipitation Temperature too low Restore the protein extraction reagent to room temperature... Read More | Product content: G665990Component200 TStorageG665990ABuffer PG100 mLRTG665990BBuffer PS60 mLRTG665990CBuffer PW (concentrate)50 mLRTG665990DBuffer EB30 mLRTG665990ESpin Columns DM with Collection Tubes200 EART Product Introduction:This kit uses a new silicon-based plasma membrane technology and Product content: G665990Component200 TStorageG665990ABuffer PG100 mLRTG665990BBuffer PS60 mLRTG665990CBuffer PW (concentrate)50 mLRTG665990DBuffer EB30 mLRTG665990ESpin Columns DM with Collection Tubes200 EART Product Introduction:This kit uses a new silicon-based plasma membrane technology and reagent formulation. Through the unique centrifugal adsorption column and the DNA washing elution step, 100 bp-10 kb DNA fragments can be recovered and purified from ordinary or low melting point agarose gel. The sol speed is fast and the recovery rate is high. The sol solution contains a pH indicator, which can be used to determine whether the sol recovery has reached the optimal state based on its color. Each adsorption column can adsorb up to 10 µ G DNA, while effectively removing impurities such as primers, enzymes, mineral oil, and agarose. The purified and recovered DNA has high purity and concentration, good integrity, and can be directly used for molecular biology experiments such as sequencing, linking and transformation, labeling, and in vitro transcription.Self prepared reagents: anhydrous ethanol, isopropanol.Preparation and important precautions before the experiment:1.Before the first use, anhydrous ethanol should be added to the Buffer PW according to the instructions on the reagent bottle label.2. Before use, please check the Buffer PG. If crystallization or precipitation occurs, it can be left in a 37 ℃ water bath for 3-5 minutes to restore clarity.3. It is best to use a new electrophoresis buffer during electrophoresis to avoid affecting the electrophoresis and recovery efficiency; The following experiment requires high requirements, please use TAE electrophoresis buffer as much as possible.4.When cutting glue, the UV irradiation time should be as short as possible to avoid damage to DNA.5. The recovery rate is related to the initial amount of DNA and the elution volume. The smaller the initial amount, the smaller the elution volume, and the lower the recovery rate.6. Preheat the water bath to 50 ℃.7. Buffer PG contains a pH indicator. When the pH is ≤ 7.5, the color of the solution is yellow, and DNA can effectively bind to the membrane. When the pH is too high, the color of the solution turns orange red and purple, which needs to be adjusted.8. All centrifugation steps can be performed at room temperature.Operation steps:1. Cut the single purpose DNA strip from the agarose gel (try to cut the excess), put it into a clean centrifuge tube (self prepared), and weigh and calculate the weight of the gel (record the weight of the centrifuge tube in advance).Attention: If the volume of the adhesive block is too large, it can be cut into small pieces.2. Add one time of the volume of Buffer PG (if the gel weighs 100 mg, its volume can be regarded as 100 µ l. And so on.3.50 ℃ water bath and gently invert the centrifuge tube every 2-3 minutes until the sol turns yellow to ensure full dissolution of the gel block. If there are still unsolved glue blocks, you can add some more sol solution or continue to let it stand for a few minutes until the glue blocks are completely dissolved.Note: 1) After the gel is completely dissolved, the gel solution is yellow, and subsequent operations can be carried out; If the glue solution is orange red or purple, 10-30 can be added to the glue solution µ 3 M sodium acetate (pH 5.0), adjust the color of the solution to yellow before proceeding with subsequent operations.2) After the gel block is completely dissolved, it is best to lower the temperature of the gel solution to room temperature before loading the column. The adsorption column has a weaker ability to bind DNA at higher temperatures.4. (Optional step) When the recovered fragment is less than 300 bp, add 1/2 of the gel volume of isopropanol, and mix it upside down (if the gel weighs 100 mg, add 50 µ Isopropanol of L.5. Column balance: Add 200 to the spin columns DM that have been loaded into the collection tube µ Centrifuge at 13000 rpm (~16200 × g) for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.6. Add the solution obtained from steps 3 or 4 to the adsorption column that has been loaded into the collection tube, let it stand at room temperature for 2 minutes, centrifuge at 13000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube.Attention: The volume of the adsorption column is 750 µ l. If the sample volume is greater than 750 µ L can be added in batches.7. Add 450 to the adsorption column µ LBuffer PW (please check if anhydrous ethanol has been added before use), centrifuge at 13000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube.Note: If purified DNA is used for salt sensitive experiments (such as flat end ligation or direct sequencing), it is recommended to add Buffer PW and let it stand for 2-5 minutes before centrifugation.8. Repeat step 7.9.13000 rpm for 1 minute and discard the waste liquid from the collection tube.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).10. Place the adsorption column into a new 1.5 ml centrifuge tube (provided by oneself), and add 50 drops to the middle position of the adsorption membrane in the air µ L Buffer EB, leave at room temperature for 2 minutes. Centrifuge at 13000 rpm for 1 minute and collect DNA solution- Store DNA at 20 ℃.Attention:1) To improve the recovery of DNA, the solution obtained by centrifugation can be re dropped onto the adsorption column, left at room temperature for 2 minutes, and centrifuged at 13000 rpm for 1 minute.2) The elution volume should not be less than 30 µ l. A small volume will affect the recovery efficiency.3) When recovering DNA fragments larger than 10 kb, Buffer EB should be preheated in a 50 ℃ water bath to increase recovery efficiency.Note: This reagent kit is also suitable for the purification and recovery of PCR products. Add an equal volume of Buffer PG to the PCR reaction solution and mix thoroughly (for small fragments with a recovery of less than 150bp, the solution volume can be increased to three times to improve the recovery rate). Follow step 5 above for further operations... 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 |