| Description | Pyruvate Kinase (PK) is widely present in animals, plants, microorganisms, and cultured cells. It catalyzes the final step of glycolysis, serving as one of the key rate-limiting enzymes in this process and a crucial enzyme for ATP production. Therefore, determining PK activity is of significant Pyruvate Kinase (PK) is widely present in animals, plants, microorganisms, and cultured cells. It catalyzes the final step of glycolysis, serving as one of the key rate-limiting enzymes in this process and a crucial enzyme for ATP production. Therefore, determining PK activity is of significant importance.Assay PrinciplePK catalyzes the conversion of Phosphoenolpyruvate (PEP) and ADP to ATP and Pyruvate. Lactate Dehydrogenase (LDH) further catalyzes the reaction between NADH and Pyruvate to produce Lactate and NAD⁺. The rate of decrease in NADH absorbance at 340 nm is measured, which reflects PK activity.Component50TStorageExtraction Buffer60 mL2-8℃Reagent A50 mL2-8℃Reagent B 2EA-20℃Reagent C25 µL×22-8℃Required Materials and Equipment (Not Provided)UV spectrophotometer, benchtop centrifuge, water bath, adjustable pipettes, 1 ml quartz cuvette, mortar and pestle, ice, and distilled water.Sample Preparation1.Bacteria or Cultured Cells:Collect bacteria or cells into a centrifuge tube, centrifuge, 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/bacteria).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 Procedure1.Preheat the spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. Zero the instrument with distilled water.2.Sample Assay:(1) Preparation of Working Reagent II (WR II): Just before use, dissolve the contents of one vial of Reagent B in 22.5 ml of Reagent A and 2.65 ml distilled water. Mix thoroughly. Incubate WR II at 37°C (for mammalian samples) or 25°C (for other species) in a water bath for 5 minutes. Prepare fresh for each use.(2) Preparation of Working Reagent III (WR III): Just before use, dissolve the contents of one tube of Reagent C in 1.5 ml distilled water. Mix thoroughly. Prepare fresh for each use.(3) Reaction Setup: In a 1 ml quartz cuvette, add:50 µl sample50 µl WR III900 µl WR IIMix thoroughly and immediately record the absorbance (A₁) at 340 nm at 20 seconds. Record the absorbance again (A₂) after 2 minutes and 20 seconds (140 seconds total). Calculate ΔA = A₁ - A₂.PK Activity CalculationGeneral Formula:PK Activity = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ ÷ Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ TWhere:Vₜₒₜₐₗ = Total reaction volume = 0.000975 L (975 µl)ε = NADH molar extinction coefficient = 6220 L/mol/cmd = Light path length = 1 cm10⁹ = Conversion factor from moles to nanomoles (nmol)T = Reaction time = 2 minVₛₐₘₚₗₑ = Sample volume added to reaction = 0.050 mlVₛₐₘₚₗₑₜₒₜₐₗ = Total volume of extract used = 1 ml (for tissues/cells)Cpr = Sample protein concentration (mg/ml)W = Sample mass (g)500 = Cell/Bacteria count (in millions, for the example calculation: 5 million = 500 × 10⁴)1. 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:PK Activity (nmol/min/ml) = [ΔA × 0.000975 ÷ (6220 × 1) × 10⁹] ÷ 0.050 ÷ 2Simplified Formula: PK (nmol/min/ml) = 2613 × ΔA2. For Tissues, Bacteria, or Cells:a. 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:PK Activity (nmol/min/mg prot) = [ΔA × 0.000975 ÷ (6220 × 1) × 10⁹] ÷ (0.050 × Cpr) ÷ 2Simplified Formula: PK (nmol/min/mg prot) = 2613 × ΔA ÷ Cprb. Based 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 tissue.* Calculation:PK Activity (nmol/min/g fresh weight) = [ΔA × 0.000975 ÷ (6220 × 1) × 10⁹] ÷ (W × 0.050 / 1) ÷ 2Simplified Formula: PK (nmol/min/g fresh weight) = 2613 × ΔA ÷ Wc. Based 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 (using the example of 5 million cells extracted in 1 ml):PK Activity (nmol/min/10⁴ cell) = [ΔA × 0.000975 ÷ (6220 × 1) × 10⁹] ÷ (5 × 0.050 / 1) ÷ 2Simplified Formula: PK (nmol/min/10⁴ cell) = 5.226 × ΔA... Read More | Product Content D669986Component50 TStorageD669986ABuffer SA15 mLRTD669986B2×PCR MasterMix1 mL-20℃. Avoid freeze/thaw cycle.D669986CProteinase K12.5 mgRTD669986DProteinase K Storage Buffer1.25 mLRTProductsThis kit adopts a unique buffer system containing all the reagents for rapid Product Content D669986Component50 TStorageD669986ABuffer SA15 mLRTD669986B2×PCR MasterMix1 mL-20℃. Avoid freeze/thaw cycle.D669986CProteinase K12.5 mgRTD669986DProteinase K Storage Buffer1.25 mLRTProductsThis kit adopts a unique buffer system containing all the reagents for rapid preparation of genomic DNA and PCR amplification, and is suitable for one-step extraction of genomic DNA from various plant and animal tissues and bacteria and for PCR amplification. The whole extraction process does not require liquid nitrogen grinding, organic solvent extraction, anhydrous ethanol precipitation, and the quality of extracted DNA is stable. The 2×PCR MasterMix provided in this kit is a highly compatible PCR reagent that can amplify DNA samples efficiently and specifically, which includes DNA polymerase, dNTPs, MgCl2, reaction buffer, PCR reaction enhancer and so on. It is characterized by fast and easy, high sensitivity, high specificity, good stability, etc. It is especially suitable for high throughput screening.Pre-experiment Preparation and Important Notes1. Add the specified amount of Proteinase K Storage Buffer to Proteinase K to dissolve it and store it at -20℃. Do not leave the prepared Proteinase K at room temperature for a long time, and avoid repeated freezing and thawing to avoid affecting its activity.2. Repeated freezing and thawing of the samples should be avoided, as this will result in smaller DNA fragments and a decrease in the amount of extracted DNA.3. Before use, please check Buffer SA for crystallization or precipitation. If crystallization or precipitation occurs, please re-dissolve Buffer SA in a 56℃ water bath.4. The PCR MasterMix provided with this product is 2×, when using it, you need to add template and primer, and add RNase-Free Water to make up the volume, so that its concentration is 1× to carry out the reaction.Procedure1. Fetch:Plant material: take about 10 mg of sample in a centrifuge tube (provided); Animal material: take about 10 mg of sample in a centrifuge tube (provided);Bacteria: Take 200-800 µL of bacteria in good growth condition in a centrifuge tube (self-provided) and collect the bacteria.2. Add 200 µL of Buffer SA and vortex to mix.Note: In the case of plant leaves and animal tissues, they should be ground with a pestle and mortar as much as possible: in the case of plant seeds, they should be crushed and finely ground beforehand; bacterial and 1-3 mm rat-tail samples can be directly vortex lysed.3. Add 10µL of Proteinase K, mix well, incubate at 56℃ for 10 minutes, and treat at 95℃ for 5 minutes.Note: 1) In the case of animal tissue samples, the incubation time at 56°C may be extended to 30 minutes as appropriate; if there is any incompletely digested tissue, it should be removed as thoroughly as possible after centrifugation in the next step.2) Be careful not to exceed 5 minutes when treating at 95°C.4. 13,000 rpm (~17,900 x g), centrifugation for 5 minutes.5. Transfer the supernatant to a new centrifuge tube (self-prepared) and use it directly for PCR amplification, or store the solution at 4℃ or -20℃.6. PCR amplification:1) PCR reaction system:The following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.reagents20 µL systemfinal concentration2×PCR MasterMix10 µL1×Forward Primer, 10 µM1 µL0.4 µMReverse Primer, 10 µM1 µL0.4 µMTemplate DNA1-2 µL RNase-free Waterup to 20 µLNote: Please use the final concentration of 0.2-0.6µM as a reference for setting the range of primer concentration. If the amplification efficiency is not high, the concentration of primer can be increased; if a non-specific reaction occurs, the concentration of primer can be decreased, thus optimizing the reaction system.2）PCR reaction conditions:movetemptimingpremutability94°C2mindenaturation94°C30sannealing (metallurgy)55-65°C30s30-40 cyclesreach72°C60sultimate extension72°C5minNote: 1) In general, the annealing temperature is 5℃ lower than the melting temperature of the amplification primer Tm, and the annealing time is generally 30-60 seconds. When the desired amplification efficiency cannot be obtained, the annealing temperature should be lowered appropriately; when a non-specific reaction occurs, the annealing temperature should be raised, thus optimizing the reaction conditions.(2) The extension time is set according to the size of the fragment to be amplified, and the amplification efficiency of Taq DNA Polymerase included in this product is 1kb/30s. 3) The number of cycles can be set according to the downstream application of the amplification product. If the number of cycles is too low, the amplification is insufficient; if the number of cycles is high, the chance of mismatch will increase and the non-specific background will be serious. Therefore, the number of cycles should be minimized under the premise of ensuring the product yield.(3) Result detection: 5 µL of reaction product was taken at the end of the reaction and directly detected by agarose gel electrophoresis... 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 | 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 | Inquire |