| Description | Phosphoenolpyruvate Carboxykinase (PEPCK, EC 4.1.1.32) is widely present in animals, plants, microorganisms, and cells. It catalyzes the conversion of oxaloacetate to phosphoenolpyruvate and is a key regulatory enzyme in the gluconeogenesis pathway.Assay PrinciplePEPCK catalyzes the conversion of Phosphoenolpyruvate Carboxykinase (PEPCK, EC 4.1.1.32) is widely present in animals, plants, microorganisms, and cells. It catalyzes the conversion of oxaloacetate to phosphoenolpyruvate and is a key regulatory enzyme in the gluconeogenesis pathway.Assay PrinciplePEPCK catalyzes the conversion of Oxaloacetate to Phosphoenolpyruvate and CO₂. Pyruvate Kinase and Lactate Dehydrogenase subsequently catalyze the sequential oxidation of NADH to NAD⁺. The rate of decrease in NADH absorbance at 340 nm is measured, which reflects PEPCK activity.Component100TStorageExtraction Buffer100 mL2-8℃Reagent 118 mL2-8℃Reagent 216.5 µL2-8℃Reagent 31EA-20℃Reagent 41EA-20℃Required Materials and Equipment (Not Provided)Spectrophotometer / Microplate reader, benchtop centrifuge, adjustable pipettes, micro quartz cuvette / 96-well plate, mortar and pestle, ice, and distilled water.Sample Preparation:1.Bacteria or Cultured Cells:Collect cells by centrifugation and discard the supernatant.Add Extraction Buffer at a ratio of 1 ml per 5-10 million cells (e.g., 1 ml for 5 million cells).Sonicate on ice (20% power or 200W, pulse 3s on/10s off, repeat 30 times).Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.2.Tissues:Homogenize tissue on ice in Extraction Buffer at a ratio of 1:5-10 (w/v) (e.g., 0.1 g tissue in 1 ml buffer).Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.3.Serum (or Plasma) Samples:Assay directly.Assay Procedure:1.Preheat the spectrophotometer or microplate reader for at least 30 minutes. Set the wavelength to 340 nm. Zero the instrument with distilled water.2.Preparation of Working Solution: Just before use, transfer and dissolve Reagent 2 and Reagent 3 into Reagent 1. Mix well. Aliquot and store any unused portions at -20°C. Avoid repeated freeze-thaw cycles.3.Preparation of Reagent 4: Just before use, dissolve the contents of the vial in 1 ml of distilled water. Mix well. Aliquot and store any unused portions at -20°C. Avoid repeated freeze-thaw cycles.4.Pre-warm the Working Solution and dissolved Reagent 4 at 37°C (for mammalian samples) or 25°C (for other species) for 5 minutes.5.In a micro quartz cuvette or a well of a 96-well plate, add:10 µl sample10 µl dissolved Reagent 4180 µl pre-warmed Working SolutionMix immediately and record the initial absorbance (A₁) at 340 nm. Record the absorbance again (A₂) after exactly 1 minute. Calculate ΔA = A₁ - A₂.Note: For this kit, if ΔA is greater than 0.1, dilute the sample with Extraction Buffer by an appropriate factor (account for this dilution factor 'n' in the calculations) so that ΔA is less than 0.1 to improve detection sensitivity.PEPCK Activity Calculation:1. Calculation for Micro Quartz Cuvette (d = 1.0 cm)General Parameters for Cuvette:Vₜₒₜₐₗ (Total reaction volume) = 0.0002 L (200 µL)ε (NADH molar extinction coefficient) = 6220 L/mol/cmd (Cuvette light path) = 1.0 cmVₛₐₘₚₗₑ (Sample volume in reaction) = 0.01 mL (10 µL)T (Reaction time) = 1 minVₛₐₘₚₗₑₜₒₜₐₗ (Total extract volume) = 1 mL (for tissues/cells)Cpr (Sample protein concentration, mg/mL)W (Sample mass, g)500 (Cell/Bacteria count in millions for example calculation: 5 million)a. For Serum (Plasma):Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per ml of serum.Calculation:PEPCK Activity (nmol/min/ml) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ Vₛₐₘₚₗₑ ÷ TSimplified Formula: PEPCK (nmol/min/ml) = 3215 × ΔAb. For Tissues, Bacteria, or Cells:Based on Sample Protein Concentration:Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per mg of protein.Calculation:PEPCK Activity (nmol/min/mg prot) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ × Cpr) ÷ TSimplified Formula: PEPCK (nmol/min/mg prot) = 3215 × ΔA ÷ CprBased on Sample Fresh Weight:Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per gram of fresh tissue.Calculation:PEPCK Activity (nmol/min/g fresh weight) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (W × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ TSimplified Formula: PEPCK (nmol/min/g fresh weight) = 3215 × ΔA ÷ WBased on Bacterial or Cell Density:Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per 10⁴ cells.Calculation (example for 5 million cells in 1 ml extract):PEPCK Activity (nmol/min/10⁴ cell) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (500 × Vₛₐₘₚₗₑ / Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ TSimplified Formula: PEPCK (nmol/min/10⁴ cell) = 6.43 × ΔA2. Calculation for 96-Well Plate (d = 0.5 cm)General Parameters for 96-Well Plate:(All parameters remain the same except for the light path 'd')d (96-well plate light path) = 0.5 cma. For Serum (Plasma):Simplified Formula: PEPCK (nmol/min/ml) = 6430 × ΔAb. For Tissues, Bacteria, or Cells:Based on Sample Protein Concentration:Simplified Formula: PEPCK (nmol/min/mg prot) = 6430 × ΔA ÷ CprBased on Sample Fresh Weight:Simplified Formula: PEPCK (nmol/min/g fresh weight) = 6430 × ΔA ÷ WBased on Bacterial or Cell Density:Simplified Formula: PEPCK (nmol/min/10⁴ cell) = 12.86 × ΔAPrecautionsBefore formal assay, it is essential to perform a pilot test with 2-3 samples expected to have significant differences in activity... 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 | DescriptionMetathesis: Ruthenium-Based Metathesis CatalystsRuthenium metathesis catalysts kit I consists of 9 samples of Grubbs 1st and 2nd generation catalysts. These catalysts have applications in ring-closing and ring-opening metathesis, cross-metathesis, ring-opening metathesis polymerization (DescriptionMetathesis: Ruthenium-Based Metathesis CatalystsRuthenium metathesis catalysts kit I consists of 9 samples of Grubbs 1st and 2nd generation catalysts. These catalysts have applications in ring-closing and ring-opening metathesis, cross-metathesis, ring-opening metathesis polymerization (ROMP) and enyne metathesis.Metathesis: Ruthenium-Based Metathesis Catalysts... Read More | Product contentS665868Component50 TStorageS665868ABuffer GL25 mLRTS665868BBuffer GW1 (concentrate)13 mLRTS665868CBuffer GW2 (concentrate)15 mLRTS665868DBuffer GE15 mLRTS665868EProteinase K2×1.25 mLRTS665868FSpin Columns DM with Collection Tubes50 setsRTProduct IntroductionThis kit is suitable Product contentS665868Component50 TStorageS665868ABuffer GL25 mLRTS665868BBuffer GW1 (concentrate)13 mLRTS665868CBuffer GW2 (concentrate)15 mLRTS665868DBuffer GE15 mLRTS665868EProteinase K2×1.25 mLRTS665868FSpin Columns DM with Collection Tubes50 setsRTProduct IntroductionThis kit is suitable for the extraction of genomic DNA from fresh saliva or saliva/preservation solution mixture.The purification process of this product does not require the use of toxic solvents such as phenol or chloroform, and ethanol precipitation is not necessary. The optimized buffer system enables DNA to bind heterogeneously to the silica matrix centrifugal adsorption column, and the inhibitors of PCR and other enzymatic reactions can be effectively removed by a two-step washing step, and finally eluted with a low-salt buffer or water to obtain high-purity DNA.The purified obtained can be directly used for enzyme digestion, PCR, Real-Time PCR, library construction, Southern Blot, molecular labeling and other downstream experiments.Self-contained reagent: anhydrous ethanolPre-experiment Preparation and Important Notes1. Repeated freezing and thawing of the sample should be avoided, as this may result in smaller fragments of extracted DNA and a decrease in the amount extracted.2. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the label of the reagent bottle before first use.3. Before use, please check whether Buffer GL appears to be crystallized or precipitated.Redissolve in a 56°C water bath.4. If the downstream experiments are sensitive to RNA contamination, 4 µL DNase-Free RNase A can be added in step 3(100 mg/mL).5. For prolonged storage of salivary DNA at room temperature, our Salivary DNA Preservation Tubes are recommended.Operation steps1. Add 400 µL of saliva sample or saliva/preservation solution mixture.Note: 1) Saliva mixtures added to the preservation solution require a 50°C water bath for 1 hour or an empty 50°C temperature chamber for 2 hours prior to extraction.2) If an increase in sample volume is required, multiply the volumes of Proteinase K, Buffer GL, and anhydrous ethanol in Steps 2-4, and the liquid can be transferred in multiple times in Step 5.2. Add 40 µL of Proteinase K.3. Add 400µL Buffer GL, vortex and shake to mix thoroughly, and water bath at 56℃ for 15-30 minutes.Note: If RNA removal is required, add 4 µL of RNase A solution at a concentration of 100 mg/mL after the above steps are completed, vortex for 15 seconds, and leave at room temperature for 2 minutes.4. Centrifuge briefly to remove water droplets from the inside of the tube cap. Add 400 µL of anhydrous ethanol and mix well by vortexing and shaking. Centrifuge briefly.Note: 1) Vortex and shake to mix immediately after adding Buffer GL and anhydrous ethanol.The addition of Buffer GL and anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.2) A sol-gel product may be formed after GL and anhydrous ethanol, in which case vigorous shaking or vortexing is recommended.3) The solution obtained in the previous step is added to the adsorption column in the Collection Tube.5. (Spin Column DM) in the collection tube, and if the solution cannot be added all at once, it can be transferred in several times. centrifuge at 12,000 rpm (∼13,400 × g) for 1 min, pour off the waste solution in the collection tube, and put the adsorption column back into the collection tube.6. Add 500 µL of Buffer GW1 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.7. Add 500 µL of Buffer GW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.Note: Step 7 can be repeated if further DNA purity is required.8. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Leave the adsorption column at room temperature for several minutes to dry thoroughly.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).9. Place the adsorption column in a new centrifuge tube (supplied), add 50-200 µL of Buffer GE or sterilized water to the middle of the adsorption column overhanging the column, let it stand at room temperature for 2-5 minutes, and centrifuge at 12,000 rpm for 1 minute to collect the DNA solution.-20°C to preserve DNA.Note: 1) If the downstream experiment is sensitive to pH or EDTA, you can use sterilized water for elution. The pH of the eluent has a great influence on the elution efficiency, if water is used as the eluent should ensure that its pH is 7.0-8.5 (you can use NaOH to adjust the pH of the water to this range), and the elution efficiency is not high when the pH is lower than 7.0.2) Buffer GE preheated in a 65-70°C water bath and incubated at room temperature for 5 min before centrifugation can increase the yield.3) Because DNA preserved in water is subject to acidic hydrolysis, for long-term storage, elution with Buffer GE and storage at -20°C is recommended... Read More | The Succinic Acid (Succinate) assay kit is suitable for the specific assay of succinic acid in wine, cheese, eggs, sauce and other food products. Succinic acid (or succinate) is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the The Succinic Acid (Succinate) assay kit is suitable for the specific assay of succinic acid in wine, cheese, eggs, sauce and other food products. Succinic acid (or succinate) is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the Citric Acid Cycle. Succinic acid concentrations are monitored in the manufacture of numerous foodstuffs and beverages, including wine, soy sauce, soy bean flour, fruit juice and dairy products (e.g. cheese).Product Description： Succinic acid is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the Citric Acid Cycle. Succinic acid concentrations are monitored in the manufacture of numerous foodstuffs and beverages, including wine, soy sauce, soy bean flour, fruit juice and dairy products (e.g. cheese). The ripening process of apples can be followed by monitoring the falling levels of succinic acid. The occurrence of > 5 mg/kg of this acid in egg and egg products is indicative of microbial contamination. Apart from use as a flavouring agent in the food and beverage industries, succinic acid finds many other non-food applications, such as in the production of dyes, drugs, perfumes, lacquers, photographic chemicals and coolants. Preparation Instructions：Suitable for succinate determination in food, beverage, agricultural products, and other biological samples.Note for Content：The number of manual tests per kit can be doubled if all volumes are halved. This can be readily accommodated using the MegaQuantTM Wave Spectrophotometer (D-MQWAVE).Browse all of our organic acid assay kits.Principle：The Succinate Assay Kit provides a simple, one step assay for measuring succinate. In this assay succinate is converted to pyruvate which reacts with specific reagents and dye to form a colored product. The color intensity at 570 nm or fluorescencAdvantages：Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.Very competitive price (cost per test)All reagents stable for > 2 years as suppliedVery rapid reaction (even at room temperature)Mega-Calc™ software tool is available from our website for hassle-free raw data processingStandard includedSuitable for manual, microplate and auto-analyser formats... Read More |