| Description | IntroductionCreatine Kinase (CK) is primarily found in tissues such as the heart, muscle, and brain. It reversibly catalyzes the transphosphorylation reaction between creatine and ATP, playing a vital role in energy transfer, muscle contraction, and ATP regeneration. It is a crucial clinical IntroductionCreatine Kinase (CK) is primarily found in tissues such as the heart, muscle, and brain. It reversibly catalyzes the transphosphorylation reaction between creatine and ATP, playing a vital role in energy transfer, muscle contraction, and ATP regeneration. It is a crucial clinical indicator for diagnosing heart and brain diseases.Assay PrincipleCK catalyzes the conversion of Phosphocreatine and ADP to Creatine and ATP. Hexokinase then catalyzes the reaction of ATP with Glucose to form Glucose-6-Phosphate (G6P). Subsequently, Glucose-6-Phosphate Dehydrogenase (G6PDH) catalyzes the oxidation of G6P with NADP⁺ to generate NADPH, leading to an increase in absorbance at 340 nm.Component50TStorageExtraction Buffer60 mL2-8℃Reagent 11EA2-8℃. Store in the dark.Reagent 215 mL2-8℃Reagent 1: Powder in one bottle. Store at 4°C protected from light. Dissolve in 15 mL distilled water before use.Working Solution: Prepare immediately before use by mixing the dissolved Reagent 1 and Reagent 2 at a 1:1 ratio. Incubate the Working Solution at 37°C for 2 minutes prior to use.Required Materials and Equipment (Not Provided)Balance, refrigerated centrifuge, constant temperature water bath, UV spectrophotometer, 1 ml quartz cuvette, and distilled water.Crude Enzyme Extraction:Tissue Samples: Homogenize the tissue on ice in Extraction Buffer at a ratio of 1:5-10 (w/v) (e.g., weigh ~0.1g tissue, add 1 mL Extraction Buffer). Centrifuge the homogenate at 10,000 g, 4°C for 15 min. Collect the supernatant and keep it on ice for assay.Serum Samples: Assay directly.Assay Procedure:Preheat the UV spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. Zero the instrument with distilled water.In a 1 ml quartz cuvette, add:200 µl sample300 µl distilled water500 µl pre-warmed (37°C) Working SolutionMix thoroughly and immediately record the initial absorbance (A₁) at 340 nm. Record the absorbance again (A₂) after exactly 1 minute at 37°C. Calculate ΔA = A₂ - A₁.CK Enzyme Activity Calculation:General Parameters:ε (NADPH molar extinction coefficient) = 6220 L/mol/cmd (Cuvette light path) = 1.0 cmVₜₒₜₐₗ (Total reaction volume) = 1.0 mL (1000 µL)Vₛₐₘₚₗₑ (Sample volume in reaction) = 0.2 mL (200 µL)T (Reaction time) = 1 minCpr (Sample protein concentration, mg/mL)W (Sample mass, g)Vₛₐₘₚₗₑₜₒₜₐₗ (Total extract volume) = Assumed 1 mL for tissue calculations1. Based on Tissue Protein Content:Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per mg of protein at 37°C, pH 7.0.Calculation:CK Activity (nmol/min/mg prot) = [ΔA / (ε × d)] × Vₜₒₜₐₗ ÷ (Vₛₐₘₚₗₑ × Cpr) ÷ TSimplified Formula: CK (nmol/min/mg prot) = 804 × ΔA ÷ Cpr2. Based on Tissue Sample Mass:Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per gram of fresh tissue at 37°C, pH 7.0.Calculation:CK Activity (nmol/min/g fresh weight) = [ΔA / (ε × d)] × Vₜₒₜₐₗ ÷ (Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ × W) ÷ TSimplified Formula: CK (nmol/min/g fresh weight) = 804 × ΔA ÷ W3. Based on Serum:Definition: One unit of activity is defined as the amount of enzyme that generates 1 nmol of NADPH per minute per liter of serum at 37°C, pH 7.0.Calculation:CK Activity (nmol/min/L) = [ΔA / (ε × d)] × Vₜₒₜₐₗ ÷ Vₛₐₘₚₗₑ ÷ TSimplified Formula: CK (nmol/min/L) = 804 × ΔANotesBefore the formal assay, it is essential to perform a pilot test with 2-3 samples expected to have significant differences in activity.The prepared Working Solution is stable at 4°C for 7 days. However, it is recommended to use it as soon as possible after preparation.CK in serum is unstable. Determine the activity as soon as possible after sample collection. It can be stored protected from light at 4°C for up to 24 hours.Sample protein content needs to be determined separately. A BCA Protein Assay Kit can be used for this purpose.If the absorbance value (ΔA) is greater than 0.5, dilute the sample appropriately with Extraction Buffer and account for the dilution factor (D) in the calculation formulas (e.g., 804 × ΔA × D ÷ Cpr)... 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 | Products contentProducts IntroductionThe Single Cell Whole Genome Amplification Kit can be used as a template for whole genome amplification of single cells or micro samples. The total time for single-cell amplification is about 3 hours, and 2-5 µg of genomic DNA, with a size of 200-1500 bp, Products contentProducts IntroductionThe Single Cell Whole Genome Amplification Kit can be used as a template for whole genome amplification of single cells or micro samples. The total time for single-cell amplification is about 3 hours, and 2-5 µg of genomic DNA, with a size of 200-1500 bp, can be obtained after lysis, pre-amplification and amplification. The amplified product can be widely used in second-generation sequencing, large fragment copy number variation analysis, SNP typing, qPCR analysis and gene chip analysis.Bring your own instruments and reagentsPCR instrument Reaction tubes: low adsorption tubes recommended Gun Heads: High quality filtered gun heads are recommended Microcentrifuge, vortex mixercaveat The sensitivity of this product is very high, the experimental operation should be completed in a positive pressure ultra-clean bench or clean environment, the concentration of the amplification reaction products is high, should be well isolated to avoid aerosol contamination caused by amplification products.Operation flow diagramprocedurePre-experiment preparationSingle cells were obtained by flow cytometry sorting, buffer dilution, micromanipulation and laser microdissection. It is recommended that the cells be washed prior to the experiments with a 1× PBS solution free of Mg2+ and Ca2+, taking care to ensure that the volume of PBS solution in subsequent experiments does not exceed 2 µl. take note of Since the whole experiment is carried out in the same PCR tube and the reaction volume is small, the pipette tip should not touch the liquid in the tube when adding liquid, so as to avoid taking single cells or DNA out of the reaction system; when pipetting, please add the liquid along the wall of the tube carefully and do not blow the liquid in the PCR tube; before the reaction, please centrifuge briefly to make sure that the liquid in the reaction system is mixed evenly. Thaw the cell lysate, pre-amplifier and amplifier on ice before use.cell lysis 1）Mix Cell Lysis Buffer and Cell Lysis Enzyme according to the number of reactions N, shake to mix, centrifuge briefly and set aside.2）Mix single cells with the cell lysis mix in a PCR tube and run the following program.2. Pre-amplification reaction1）Mix Cell Lysis Buffer and Cell Lysis Enzyme according to the number of reactions N, shake to mix, centrifuge briefly and set aside.2）Add 5 µl of pre-amplification mix to 10 µl of lysis reaction product from the previous step and run the following program. 3. Amplification reaction1）Mix Amplification Buffer and Amp Enzyme Mix according to the number of reactions N, mix with shaking, centrifuge briefly and set aside.2）Add 60 µl of amplification mix to 15 µl of pre-amplification reaction product from the previous step and run the following program.Note: The number of cycles can be adjusted as needed, 14 cycles are recommended for single cells obtained by flow sorting, etc.Amplification product detection 1. Agarose gel electrophoresis 5 µl of the amplified product was subjected to agarose gel electrophoresis (1% agarose gel, 110 V, 25-35 min), and the amplified product was 200-1500 bp in size. 2. Quantitative Amplification products were subjected to magnetic bead or column purification, and purified products were quantified using Qubit with a final yield of 2-5 µg... 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 |