| Description | Creatine 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 Creatine 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. Component100TStorageExtraction Buffer100 mL2-8℃Reagent 11EA2-8℃. Store in the dark.Reagent 210 mL2-8℃Reagent 1: Powder in one bottle. Store at 4°C protected from light. Dissolve in 10 mL distilled water before use.Working Solution: Prepare immediately before use by mixing 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, microplate reader, 96-well plate, 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 for assay.Serum Samples: assay directly.Assay Procedure:Preheat the microplate reader for at least 30 minutes. Set the wavelength to 340 nm.Pipette 40 µl of sample and 60 µl of distilled water into a well of the 96-well plate. Add 100 µl of the pre-warmed (37°C) Working Solution. Mix immediately and record the initial absorbance (A₁) and the absorbance after exactly 1 minute (A₂) at 37°C. Calculate ΔA = A₂ - A₁.CK Enzyme Activity Calculation:General Parameters:ε (NADPH molar extinction coefficient) = 6220 L/mol/cmd (Light path for 96-well plate) = 0.5 cmVₜₒₜₐₗ (Total reaction volume) = 0.2 mL (200 µL)Vₛₐₘₚₗₑ (Sample volume in reaction) = 0.04 mL (40 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) = 1608 × 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) = 1608 × 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) = 1608 × ΔANotesBefore 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 OD value 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., 1608 × ΔA × D ÷ Cpr)... 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 | DescriptionProvides an inert environment to run oxygen sensitive cross-coupling reactions in a laboratory fume hood.Designed to be used with KitAlysis High-Throughput Screening Kits and KitAlysis 24-Well Reaction Block | Product contentN666081Component50 TStorageN666081ANc-Buffer A50 mL2-8℃N666081BNc-Buffer B3 mL2-8℃N666081CNc-Buffer C25 mL2-8℃N666081DProtease Inhibitor Cocktail750 µL-20℃. Avoid freeze/thaw cycle.ProductsThe Nc-Nucleus/Plasma Protein Extraction Kit is a simple and rapid Product contentN666081Component50 TStorageN666081ANc-Buffer A50 mL2-8℃N666081BNc-Buffer B3 mL2-8℃N666081CNc-Buffer C25 mL2-8℃N666081DProtease Inhibitor Cocktail750 µL-20℃. Avoid freeze/thaw cycle.ProductsThe Nc-Nucleus/Plasma Protein Extraction Kit is a simple and rapid method for extracting nucleus and plasma proteins from mammalian cells and tissues, and the extracted proteins remain biologically active. The kit first cleaves the cell membrane and releases plasma proteins using the plasma protein extraction reagent, and then centrifuges the nucleus to obtain a nucleus precipitate. Finally, the nuclear proteins are extracted by the nuclear protein extraction reagent. The extracted nuclear and plasma proteins are of high purity, effectively avoiding cross-contamination of nuclear and plasma proteins, and can be used for subsequent operations such as Western, Gel Shift, reporter gene detection and enzyme activity determination.Caveat1. If phosphorylated proteins are to be extracted, add a phosphatase inhibitor to the extraction reagent.2. All sample handling should be done on ice.3. The amount of reagents can be adjusted according to the specific experimental situation to ensure that the ratio of each reagent used is Nc-Buffer A:Nc-Buffer B:Nc-Buffer C = 100:5.5:50.4. Higher speeds can be used for centrifugation.ProcedureI Extraction of cytoplasmic and cytosolic proteins from cells1. Please remove the extraction reagents Nc-Buffer A and Nc-Buffer C for pre-cooling before protein extraction.2. Collect the cells and count them. Centrifuge to remove supernatant.3. 1×107 cells were added with 1 ml of Nc-Buffer A (added to Protease Inhibitor Cocktail at a ratio of 1:99 within 2-3 minutes prior to protein pumping), vortexed for 5 seconds to mix well, and incubated on ice for 20 minutes.Note: The characteristics of various cells are different, and the amount of Nc-Buffer A needs to be adjusted according to the characteristics of different cells. If the protein concentration is small, reduce the amount of Nc-Buffer A and subsequent Nc-Buffer B and Nc-Buffer C proportionally.4. Add 55 µl of Nc-Buffer B, vortex for 5 seconds to mix thoroughly, and incubate on ice for 1 minute.5. Centrifuge at 12,000 rpm (~13,400 x g) for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is cytoplasmic protein).6. Add 500 µl of Nc-Buffer C (add Protease Inhibitor Cocktail at a ratio of 1:99 before use) to the precipitate obtained in the previous step, vortex for 5 seconds to mix thoroughly, resuspend the precipitate and incubate on ice for 40 minutes, vortexing and mixing at 10-minute intervals for about 15-30 seconds each time.7. Centrifuge at 12,000 rpm for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is for cytosolic proteins).II Extraction of cytoplasmic and cytosolic proteins from tissues1. Sampling and preservation of tissues.2. Remove the extraction reagents Nc-Buffer A and Nc-Buffer C for pre-cooling before protein extraction.3. Weigh the tissue and add 1 ml of Nc-Buffer A per 100 mg of tissue (add Protease Inhibitor Cocktail 2-3 minutes before protein extraction at a ratio of 1:99), homogenize well on ice with a homogenizer, and incubate on ice for 20 minutes.Note: The characteristics of various tissues are different, and the amount of Nc-Buffer A needs to be adjusted according to different tissues. If the protein concentration is small, reduce the amount of Nc-Buffer A and subsequent Nc-Buffer B and Nc-Buffer C proportionally.4. Add 55 µl of Nc-Buffer B, vortex for 5 seconds to mix thoroughly, and place on ice for 1 minute of incubation.5. Centrifuge at 12,000 rpm for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is cytoplasmic protein).6. Add 500 µl of Nc-Buffer C (add Protease Inhibitor Cocktail at a ratio of 1:99 before use) to the precipitate obtained in the previous step, vortex for 5 seconds to mix thoroughly, resuspend the precipitate and incubate on ice for 40 minutes, vortexing and mixing at 10-minute intervals at, each time for about 15-30 seconds.7. Centrifuge at 12,000 rpm for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is cytosolic protein)... Read More | This reagent kit uses highly sensitive silver dye, which can be applied to protein staining of denatured and non denatured gels. It has the advantages of clear target bands, low background, and flexible control of operation time. In addition, this reagent kit has added a short-term sensitization This reagent kit uses highly sensitive silver dye, which can be applied to protein staining of denatured and non denatured gels. It has the advantages of clear target bands, low background, and flexible control of operation time. In addition, this reagent kit has added a short-term sensitization step, which can significantly reduce the background and enhance the brightness of the target band. P665901Component20 TStorageP665901ASilver Stain Sensitizer (500×)2×1 mLRTP665901BSilver Stain Enhancer3 mLRTP665901CSilver Stain2×250 mLRTP665901DSilver Stain Developer4×125 mLRT Matters needing attention1. Please prepare 50 ml of fixed solution (ultrapure water: ethanol: acetic acid=6:3:1), 50 ml of eluent (10% ethanol), and 50 ml of termination solution (5% acetic acid) in advance.2. Please use deionized water and clean glass or plastic containers during operation, and wear disposable gloves for operation.The entire silver dyeing process needs to be carried out on a shaker, with a rotation speed of about 60 rpm.4. Self prepared ethanol and glacial acetic acid are required.Instructions for useThe dosage of each solution in the following operation steps takes the gel with a size of 8.5 × 5.5 cm and a thickness of 1.0 mm as an example. The gel is immersed in the solution completely, and is operated on a shaker, with a general dosage of 25 ml. For large gel, the dosage of each solution should be scaled up according to the gel volume. Please prepare 50 ml of fixed solution (ultrapure water: ethanol: glacial acetic acid=6:3:1), 50 ml of eluent (10% ethanol), and 50 ml of termination solution (5% glacial acetic acid) in advance.1. Water washing: After electrophoresis is completed, wash the gel twice with ultrapure water for 5 minutes each time.2. Fixation: Fix the gel twice with 25 ml of fixative solution for 15 minutes each time.3. Elution: Wash the adhesive twice with eluent, each time for 5 minutes.4. Water washing: Wash the glue twice with ultrapure water, each time for 5 minutes.5. Sensitization: put the gel washed in the previous step into the silver dye sensitization working solution, incubate it accurately for 1 minute at room temperature, and then wash it with ultrapure water for three times, each time for 20 seconds. Preparation of silver staining sensitization working solution: Take 50 µ l Silver Stain Sensitivity (500 x) and add it to 25 ml of ultrapure water, mix well.6. Silver staining: discard ultrapure water and incubate gel in silver staining working solution for 30 minutes. Preparation of silver staining working solution: Take 25ml Silver Stain and add 50 µ l Silver Stain Enhanced to mix well.7. Water washing: Quickly wash the glue twice with ultrapure water, with each washing accurately controlled for 20 seconds.8. Development: Immerse the washed gel in the developer immediately and incubate it at room temperature for 2-3 minutes until the protein strip is clear. Preparation of developer: Take 25ml Silver Stain Developer and add 30 µ l Silver Stain Enhanced to mix well. Attention: Within 30 seconds of development, protein bands begin to appear and continue to develop for 2-3 minutes. If the protein band appears lighter, the development time can be appropriately extended to 5 minutes or more.9. Termination: After washing the developer on the gel with the termination solution, soak the gel in a new termination solution to react for 10 minutes.Experimental imagesSilver staining results of BSA protein samples after 10% SDS-PAGE gel electrophoresisThe molecular weight of BSA protein is about 66 kD, and the loading amounts from left to right are 50 ng, 10 ng, and 5 ng, respectively... Read More |