| Description | Glycogen is a macromolecular polysaccharide composed of glucose and serves as one of the primary storage forms of sugar. It is mainly stored in the liver and muscles as reserve energy, referred to as liver glycogen and muscle glycogen, respectively. Liver glycogen regulates blood glucose Glycogen is a macromolecular polysaccharide composed of glucose and serves as one of the primary storage forms of sugar. It is mainly stored in the liver and muscles as reserve energy, referred to as liver glycogen and muscle glycogen, respectively. Liver glycogen regulates blood glucose concentration; when blood sugar rises, glycogen can be synthesized in the liver, and when blood sugar decreases, liver glycogen is broken down into glucose to supplement blood sugar. Therefore, liver glycogen is crucial for maintaining the relative balance of blood glucose. Muscle glycogen is the storage form of sugar in muscles. During strenuous exercise that consumes large amounts of blood sugar, muscle glycogen cannot be directly broken down into blood sugar but must first decompose to produce lactic acid, which circulates to the liver via the bloodstream and is converted into liver glycogen and glucose through gluconeogenesis. Detection Principle: Glycogen is extracted using a strong alkaline extraction buffer. Under strong acidic conditions, it forms a blue compound with the anthrone chromogen, which has a characteristic absorption peak at 620 nm. Within a certain concentration range, the glycogen content is linearly related to the absorbance at 620 nm. The glycogen content in the sample can be calculated based on the standard curve. Detection Range: 0.003125 - 0.25 mg/mL Sensitivity: 0.003125 mg/mL Applicable Samples: Animal tissues, bacteria, cellsG1501748Component96TStorageG1501748AExtraction Buffer120 mL2-8℃G1501748BChromogen1EA2-8℃. Store in the dark.G1501748CStandard1 mL2-8℃Note: It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.User-Provided Instruments and Consumables1.Microplate reader or visible spectrophotometer (capable of measuring absorbance at 620 nm)2.Low-temperature centrifuge, Water bath3.96-well plate or micro glass cuvettes, Adjustable pipettes and tips, EP tubes4.Deionized water, Concentrated sulfuric acidExperimental Procedure1. Reagent PreparationReagent NameReagent PreparationPrecautionsExtraction BufferReady-to-use; equilibrate to room temperature before use.Store at 4°C. Corrosive; please take protective measures during handling.ChromogenFirst, dissolve the powder in 7.2 mL of deionized water. Then slowly add 28.8 mL of concentrated sulfuric acid. Mix thoroughly after complete dissolution.Store at 4°C protected from light; valid for one week. Toxic; please take protective measures during handling.StandardStore at 4°C.2. Standard Curve SetupDilute the 1 mg/mL standard with deionized water to prepare standard solutions of 0.25, 0.1, 0.05, 0.025, 0.0125, 0.00625, and 0.003125 mg/mL as shown in the table below.No.Standard VolumeDeionized Water Volume (µL)Concentration (mg/mL)Std.1100µL of 1mg/mL3000.25Std.2160µL of Std.12400.1Std.3200µL of Std.22000.05Std.4200µL of Std.32000.025Std.5200µL of Std.42000.0125Std.6200µL of Std.52000.00625Std.7200µL of Std.62000.003125Note: A standard curve must be prepared for each experiment. Diluted standard solutions are unstable and must be used within 4 hours.3. Sample PreparationNote: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month.3.1 TissuesWeigh 0.1 g of tissue and place it in a 10 mL test tube. Add 0.75 mL of Extraction Buffer. Boil in a water bath for 20 minutes (stopper the tube tightly to prevent water evaporation). Shake the tube every 5 minutes to mix thoroughly. After the tissue is completely dissolved, remove the tube and let it cool. Dilute to 5 mL with deionized water, mix well. Centrifuge at 8,000 g, 25°C for 10 minutes. Collect the supernatant for detection.3.2 Cells or BacteriaCollect 5 million bacteria or cells into an EP tube. Centrifuge and discard the supernatant. Add 0.75 mL of Extraction Buffer and disrupt the bacteria or cells by ultrasonication (power 200 W, ultrasonicate for 3 s, interval 10 s, repeat 30 times). Transfer to a 10 mL test tube. Boil in a water bath for 20 minutes (stopper the tube tightly to prevent water evaporation). Shake the tube every 5 minutes to mix thoroughly. Remove the tube and let it cool. Dilute to 5 mL with deionized water, mix well. Centrifuge at 8,000 g, 25°C for 10 minutes. Collect the supernatant for detection.Note: For protein concentration determination, Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.4. Assay Steps4.1 Instrument Preparation: Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 620 nm. For visible spectrophotometers, zero the instrument with deionized water.4.2 Sample Assay: Add reagents sequentially to EP tubes as follows:ReagentBlank Tube (µL)Standard Tube (µL)Test Tube (µL)Sample0060Standard0600Deionized Water6000Chromogen2402402404.3 Mix well. Incubate in a 95°C water bath for 10 minutes (cap tightly to prevent evaporation). Cool. Transfer 200 µL to a 96-well plate or micro glass cuvette. Measure the absorbance at 620 nm, recorded as A blank, A standard, and A test. Calculate ΔA test = A test - A blank and ΔA standard = A standard - A blank. Note: It is recommended to perform preliminary experiments with 2-3 samples expected to have significant differences before formal testing. If ΔA test is less than 0.001, appropriately increase the sample amount. If ΔA test is greater than 1.5, dilute the sample further with deionized water (multiply the result by the dilution factor) or reduce the amount of sample used for extraction. 5. Result Calculation Note: We provide both derived and simplified calculation formulas, which are equivalent. The simplified formulas in bold are recommended as the final calculation formulas. 5.1 Standard Curve Plotting Plot the standard curve with standard concentration as the y-axis and ΔA standard as the x-axis (using concentration as the y-axis facilitates calculation). Substitute ΔA test into x to calculate y (mg/mL). 5.2 Sample Glycogen Content Calculation (1) Based on sample mass: Glycogen (mg/g) = 1.11 × (y × V sample ) ÷ (W × V sample ÷ V total ) × n = 5.55 × y ÷ W × n (2) Based on sample protein concentration: Glycogen (mg/mg prot) = 1.11 × (y × V sample ) ÷ (V sample × Cpr) × n = 1.11 × y ÷ Cpr × n (3) Based on bacterial or cell count: Glycogen (mg/10⁴) = 1.11 × (y × V sample ) ÷ (Bacterial or Cell Count × V sample ÷ V total ) × n = 5.55 × y ÷ Bacterial or Cell Count × n Parameter Description: 1.11: Constant for converting glucose content measured by this method to glycogen content (i.e., 100 µg glucose color developed with anthrone reagent is equivalent to that of 111 µg glycogen). V sample : Volume of test sample added to the reaction system, 0.06 mL. W: Sample mass, g. V total : Total volume of the sample extract, 5 mL. n: Dilution factor. Cpr: Sample protein concentration, mg/mL. Bacterial or Cell Count: In units of 10⁴ (ten thousands)6. Result PresentationTypical Standard Curve: y = 0.1746x + 0.0027, R² = 0.9961(The following data and curve are for reference only; users must establish their own standard curve based on their experiment.)Precautions1. It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.2. This product is for scientific research use only and is not intended for clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation... Read More | Description：Acetylcholinesterases (AChEs) are enzymes that hydrolyze the neurotransmitter acetylcholine (ACh) to acetate and choline. AChE is located at the synaptic cleft and functions to terminate synaptic transmission by catalyzing the breakdown of ACh allowing cholinergic neurons to Description：Acetylcholinesterases (AChEs) are enzymes that hydrolyze the neurotransmitter acetylcholine (ACh) to acetate and choline. AChE is located at the synaptic cleft and functions to terminate synaptic transmission by catalyzing the breakdown of ACh allowing cholinergic neurons to return to a resting state after activation. It is also found in membranes of red blood cells, motor and sensory fibers, muscles, nerves and central and peripheral tissues. Changes in AChE activity may result from exposure to certain insecticides, which act as cholinesterase inhibitors. Inhibitors of AChE are also used to treat certain conditions such as dementia.Acetylcholinesterase activity assay kit has been used to determine the activity of acetylcholinesterase in a rat organophosphate model and in brain tissue homogenates.Principle：Acetylcholinesterase can catalyze the hydrolysis of acetylcholine to choline, and the reaction of choline with disulfide p-nitrobenzoic acid to produce 5-merhydryl-nitrobenzoic acid (TNB). The product has a characteristic absorption peak at 412 nm, and the activity of acetylcholinesterase can be characterized by the change of light absorption valueThe Dilution Calculator EquationConcentration (start)xVolume (start)= Concentration (final)× Volume (final)This equation is commonly abbreviated as: C1V1 = C2V2... Read More | 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 DescriptionOur Glycan Sequencing Kit includes the enzymes and buffer required to sequence ten N-linked oligosaccharides.ContentsNeuraminidase from Arthrobacter ureafaciens – 80 µlBeta-Galactosidase from Streptococcus pneumoniae – 60 µlN-Acetylglucosaminidase from Product DescriptionOur Glycan Sequencing Kit includes the enzymes and buffer required to sequence ten N-linked oligosaccharides.ContentsNeuraminidase from Arthrobacter ureafaciens – 80 µlBeta-Galactosidase from Streptococcus pneumoniae – 60 µlN-Acetylglucosaminidase from Streptococcus pneumoniae) – 40 µlAlpha-Mannosidase from Jack Bean – 20 µlCore Alpha-Mannosidase from X. manihotis) – 10 µl5X Reaction buffer – 400 µlAnalysisMany methods of analysis are available, including HPLC, gel electrophoresis, HPAEC, capillary electrophoresis, and mass spectrometry. For more information on these methods, please contact us.StabilityThe Glycan Sequencing Kit is stable at least 12 months when stored properly. Several days exposure to ambient temperatures will not reduce activity.PurityAll Enzymes are tested for contaminating protease by incubating 10 µg of denatured BSA with 2 µl of enzyme at 37°C for 24 hours. SDS-PAGE analysis of the treated BSA shows no evidence of degradation.The production host strains for our recombinant enzymes have been extensively tested and do not produce any detectable glycosidases. Enzymes purified from native sources are tested for contaminating exoglycosidases The absence of exoglycosidase contaminants is confirmed by extended incubations with the corresponding pNP-glycosides... Read More | Products contentN665737Component24 T96 TStorageN665737ATPS V50 168 µL672 µL-20℃. Avoid freeze/thaw cycle.N665737B5×FA Reaction Buffer96 µL384 µL-20℃. Avoid freeze/thaw cycle.N665737CTS Buffer72 µL288 µL-20℃. Avoid freeze/thaw cycle.N665737D2×Products contentN665737Component24 T96 TStorageN665737ATPS V50 168 µL672 µL-20℃. Avoid freeze/thaw cycle.N665737B5×FA Reaction Buffer96 µL384 µL-20℃. Avoid freeze/thaw cycle.N665737CTS Buffer72 µL288 µL-20℃. Avoid freeze/thaw cycle.N665737D2×PCR Mix600 µL2×1.2 mL-20℃. Avoid freeze/thaw cycle. * This kit is suitable for human genomic DNA library construction, the starting template DNA input is 5 ng. We also have transposase library construction kits for 50 ng and 1 ng of human genomic DNA starting, in order to get a higher quality library, it is recommended to use different kits for different starting amount of DNA. Products IntroductionThis kit is developed for Illumina's high-throughput sequencing platform and provides the enzyme premix system and reaction buffer for genomic DNA library construction, including all components except PCR primers. Compared with the traditional library construction kits, this kit adopts the new transposase method for library construction, which can complete DNA fragmentation, end repair and junction reaction in one simple enzymatic reaction, significantly reducing the amount of template, reducing the number of experimental steps, and shortening the time of library construction; it adopts the high-fidelity DNA polymerase for library enrichment, and the preference-free PCR amplification can expand the coverage area of the sequence, which can be used for efficient and effective sequencing. The use of high-fidelity DNA polymerase for library enrichment and preference-free PCR amplification broadens the coverage area of the sequence and enables efficient preparation of DNA libraries for Illumina's second-generation sequencing platform. The kit is suitable for use with a starting template DNA input of 5 ng, and all reagents in the kit have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction. Product Features● DNA fragmentation and junction ligation in one step.● Ultra-fidelity amplification minimizes amplification preference.Provide your own instruments, kits and consumables1. Magnetic frame: DynaMagTM-2 is recommended.2. DNA purification and recovery kit: It is recommended to use DNA purification and recovery kit by magnetic bead method.3. Library PCR primer kit: It is recommended to use transposase method for second generation sequencing multi-sample primer kit.4. Anhydrous ethanol, deionized water (pH between 7.0 and 8.0).5. Reaction tubes: It is recommended to use low adsorption PCR tubes and 1.5 ml centrifuge tubes. Tips: It is recommended to use high quality filter tips to prevent contamination of kits and library samples. Pre-experiment Preparation and Important Notes1. Avoid repeated freezing and thawing of reagents.2. PCR products are easily contaminated due to improper operation, resulting in inaccurate results. It is recommended to isolate the PCR reaction system preparation area from the PCR product purification area, and to use special pipettes to clean the experimental areas at regular intervals.3. Bead purification: the beads should be equilibrated to room temperature before use, all operations on the beads should be carried out at room temperature, 80% ethanol should be dispensed freshly, the beads should be rinsed and dried until the surface is free of liquid reflections and has a frosted appearance, insufficient drying of the beads will cause ethanol residue that will affect the subsequent experiments, and over-drying of the beads will affect the efficiency of DNA recovery.4. The kit is suitable for human genomic DNA library construction, if the DNA sample is a PCR product, it should be ensured that its length>.500 bp, since transposases do not work on DNA ends, it is recommended to extend the PCR product by 50-100 bp at each end of the PCR product to avoid low coverage of the ends for sequencing. Sample PreparationDNA purity requirements: A260/A280 = 1.8-2.0. Sample DNA: dissolve in ultrapure water. DNA Quantification: Too much or too little DNA will affect the quality of the library. It is recommended to use Nano to test the purity of the genomic DNA and then use Qubit to test the concentration of the genome (do not use any absorbance-based assay for template quantification). Schematic diagram of DNA banking processprocedureDNA fragmentation, junction reaction 1. Add the following reagents to a 200 µl PCR tube: 2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows: inactivation reactionAfter the DNA is fragmented, the enzyme is still in a high active state, so it should be removed from the PCR instrument immediately and terminated by adding the Reaction Termination Buffer, in order to prevent the DNA from being fragmented too much and resulting in smaller library fragments.1. Add 3 µl of TS Buffer to the PCR tube containing the fragmentation product.2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Incubate at room temperature for 5 min, or if the room temperature is too low, place the reaction on a PCR instrument at 25°C with the thermal cover closed.1. Add the following reagents to a 200 µl PCR tube.2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows Selective recovery of library DNA fragmentsIt is recommended to use CombiVision Magnetic Beads DNA Purification and Recovery Kit for selective recovery of DNA fragments. When different sizes of DNA fragments are required, the amount of magnetic beads used is different, please refer to the attached table for the specific amount of magnetic beads used.(If using other brands of magnetic beads, you need to figure out the optimal amount of magnetic beads by yourself).Note: Amplification products can also be fragment length sorted and purified using the Gum Recovery Kit. If there is no special requirement for library length distribution, amplification products can also be purified directly from DNA fragments without selective recovery of DNA fragments as described on page 4 of the manual.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. Transfer the PCR products to a 1.5 ml centrifuge tube, rehydrate to 100 µl, add several volumes of magnetic beads equilibrated to room temperature, vortex for 5 seconds, and let stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, and carefully aspirate the supernatant and transfer it to a new 1.5 ml centrifuge tube.Note: Do not discard the top clear.4. Add several volumes of magnetic beads to the supernatant, vortex and shake for 5 seconds, then let stand at room temperature for 5 minutes.5. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA.Note: Do not discard the beads.6. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.7. Repeat step 6 once.8. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 20 µl of ddH2O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.9. Remove the centrifuge tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer the supernatant solution to a new centrifuge tube. Table: Suggested amount of magnetic beads for different segment selection recovery Library DNA fragment purificationWe recommend the use of the Century Magnetic Bead Method DNA Purification and Recovery Kit.1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.2. 50 µl of magnetic beads equilibrated to room temperature were added to the PCR product, vortexed and shaken for 5 seconds, and then left to stand at room temperature for 5 minutes.3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (approximately 3-5 minutes), carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA. Note: Do not discard the beads.4. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 µl of freshly prepared 80% ethanol to the centrifuge tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.Note: When adding ethanol, the liquid must not be blown directly onto the beads.5. Repeat step 4.6. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 25 µl of ddH O to solubilize.Note: Do not over-dry the beads as this may affect the elution efficiency.7. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer the supernatant solution to a new tube. Library quality controlDetermination of library concentrationIn order to obtain high-quality sequencing results, accurate quantification of DNA libraries is required, and the first recommendation is to use Real-timePCR methods are used for absolute quantification of DNA libraries. Additionally, fluorescent dye methods such as the Qubit method or the fluorescent dye picogreen method can be used; do not use quantification methods based on absorbance measurements here. The following approximate formula can be used to convert the molar concentration of the DNA library. Library fragment distributionThe prepared DNA libraries can be detected by agarose gel electrophoresis or Agilent 2100 Bioanalyzer.Range of segment length distributions... Read More |