| 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 | H665581 Component 100 T Storage H665581A gDNA Eraser 50 µL -20℃. Avoid freeze/thaw cycle. H665581B 10×gDNA Eraser Buffer 120 µL -20℃. Avoid freeze/thaw cycle. H665581C HiFiScript, 200 U/µL 100 µL -20℃. Avoid freeze/thaw cycle. H665581D 5×ScriptRT H665581 Component 100 T Storage H665581A gDNA Eraser 50 µL -20℃. Avoid freeze/thaw cycle. H665581B 10×gDNA Eraser Buffer 120 µL -20℃. Avoid freeze/thaw cycle. H665581C HiFiScript, 200 U/µL 100 µL -20℃. Avoid freeze/thaw cycle. H665581D 5×ScriptRT Buffer 500 µL -20℃. Avoid freeze/thaw cycle. H665581E Primer Mix 120 µL -20℃. Avoid freeze/thaw cycle. H665581F RNase-Free Water 2×1 mL -20℃. Avoid freeze/thaw cycle.Product IntroductionThis product is a kit for removing genomic DNA for reverse transcription. The kit removes genomic DNA in 2 minutes at 42°C. Since the reverse transcription reagent contains a component that inhibits gDNA Eraser, cDNA can be synthesized directly by reverse transcription of gDNA Eraser-treated samples.The kit is equipped with a new high-efficiency reverse transcription enzyme, HiFiScript, with novel mutation sites that dramatically increase the transcriptional activity of the enzyme, resulting in higher efficiency and yield of cDNA first-strand synthesis. The first strand of cDNA can be synthesized with higher efficiency and yield, and the first strand of cDNA can be synthesized from pg total RNA or mRNA. If the reverse transcription product cDNA is used for downstream fluorescence quantitative detection, the reverse transcription reaction can be completed at 42℃ in 15 minutes. This kit is suitable for the synthesis of first-strand cDNA and subsequent RT-PCR, RT-qPCR, and the construction of full-length cDNA libraries.Product Features1. Rapid genome removal: contains gDNA Eraser for genomic DNA removal, which removes genomic DNA in just 2 minutes.2. Rapid reverse transcription: 15 minutes to obtain fluorescent quantitative PCR template cDNA first strand synthesis.3. High sensitivity: cDNA first strand can be synthesized using pg-level total RNA or mRNA templates.4. Highly efficient reverse transcription: Novel mutation sites dramatically increase enzyme activity, resulting in higher yields of cDNA.matters needing attention1. During operation, RNase contamination should be avoided to prevent RNA degradation or cross-contamination in the experiment. It is recommended that operators wear masks and disposable gloves and change the gloves frequently, and use specialized instruments and consumables.2. The reverse transcription system is prepared and operated on ice to prevent degradation of RNA. Store the kit enzymes at -20ºC as soon as possible after use and try to avoid repeated freezing and thawing.3. The reaction system can be scaled up to a maximum of 1 µg of total RNA in 10 µl of reaction system.4. Primer Mix is prepared by Oligo(dT) and Random primer, and Oligo-dT Primer or Gene Specific Primer can also be used according to the experimental needs.5. If the amount of starting RNA is less than 50ng, it is recommended to add RNAase inhibitor (RNasin).6. For RNA templates with complex secondary structures, it is recommended to incubate the template RNA at 65°C for 5 minutes immediately on ice prior to the manipulation step and centrifuge briefly before proceeding to the next step.UsageThaw template RNA on ice; place kit components on ice immediately after thawing at room temperature. Each solution was mixed by vortexing and shaking before use and briefly centrifuged.I. Genomic DNA removal reactions1. Prepare the reaction system according to the following table on ice in a total volume of 10 µl. To ensure the accuracy of the reaction solution preparation, prepare the premixed system in the amount of reaction number + 2 before dispensing it into each reaction tube and finally adding the RNA sample.Note: 1) If the amount of total RNA is greater than 1µg, scale up the reaction system proportionally. If the amount of starting RNA is less than 50ng, it is recommended to add RNAase inhibitor (RNasin).2. Mix by vortex shaking and centrifuge briefly so that the solution on the walls of the tube collects at the bottom.3. Incubate at 42°C for 2 minutes (this can be extended to 30 minutes for room temperature reactions).4．At the end of the reaction, centrifuge briefly and place on ice to cool.II. Reverse transcription reaction1. Prepare the reaction system on ice according to the following table. In order to ensure the accuracy of the reaction solution configuration, first prepare a premixed solution in the amount of number + 2, and then dispense 10 µl into each reaction tube, take 10 µl of the prepared premixed solution and add it to the reaction tube of step 1 where the de-etching of the genome has been completed.Note: 1) Oligo-dT Primer or Gene Specific Primer can be used according to the needs of the experiment, it is recommended to use 50 pmol of Oligo-dT Primer or 2 pmol of Gene Specific Primer for 20 µl reaction system.2. Mix well and centrifuge briefly so that the solution on the walls of the tube collects at the bottom.3. cDNA synthesis reaction conditions:1) If fluorescent quantitative PCR assay is performed downstream, incubate at 42°C for 15 minutes and 85°C for 5 minutes.2) If downstream for normal PCR assay, incubate at 42°C for 30-50 minutes and 85°C for 5 minutes. Note: For templates with complex secondary structure or high GC content, the reverse transcription temperature can be increased to 50°C to enhance reverse transcription efficiency.4. At the end of the reaction, centrifuge briefly and place on ice before proceeding with subsequent PCR or fluorescence quantitative PCR, or place at -20°C if prolonged storage is required.Note: When performing Real-time PCR reactions, the amount of reverse transcription product added should not exceed 1/10 of the total volume of the PCR reaction... Read More | Inquire | 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 | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export |