| 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.Component50TStorageAcidic Extraction Buffer60 mL2-8℃Reagent 145 mL2-8℃Reagent 241 µL2-8℃Reagent 31EA-20℃Reagent 41EA-20℃Required Materials and Equipment (Not Provided)UV spectrophotometer, benchtop centrifuge, adjustable pipettes, 1 ml quartz cuvette, mortar and pestle, ice, and distilled water.Sample Preparation:*Note: The provided sample-to-buffer ratios (1:1, w/v or based on cell count) using microliters (µl) are highly unusual and likely a typo in the original text. Standard protocols use milliliters (ml). The calculations also imply ml. The following protocol assumes the intended volumes are in milliliters (ml).*Bacteria or Cultured Cells:Collect cells by centrifugation and discard the supernatant.Add Acidic 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.Tissues:Homogenize tissue on ice in Acidic 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.Serum (or Plasma) Samples:Assay directly.Assay Procedure:Preheat the spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. Zero the instrument with distilled water.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.Preparation of Reagent 4: Just before use, dissolve the contents of the vial in 2.5 mL of distilled water. Mix well. Aliquot and store any unused portions at -20°C. Avoid repeated freeze-thaw cycles.Pre-warm the Working Solution and dissolved Reagent 4 at 37°C (for mammalian samples) or 25°C (for other species) for 5 minutes.In a 1 ml quartz cuvette, add:50 µl sample50 µl dissolved Reagent 4900 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 Acidic 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:General Parameters for 1 ml Cuvette (d = 1.0 cm):Vₜₒₜₐₗ (Total reaction volume) = 0.001 L (1000 µL)ε (NADH molar extinction coefficient) = 6220 L/mol/cmd (Cuvette light path) = 1.0 cmVₛₐₘₚₗₑ (Sample volume in reaction) = 0.05 mL (50 µL) [Note: Corrected from 0.05µL, which is implausible]T (Reaction time) = 1 minVₛₐₘₚₗₑₜₒₜₐₗ (Total extract volume) = 1 mL (for tissues/cells) [Note: Corrected from 1µL]Cpr (Sample protein concentration, mg/mL) [Note: Corrected from mg/µL]W (Sample mass, g)500 (Cell/Bacteria count in millions for example calculation: 5 million)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:PEPCK Activity (nmol/min/ml) = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ Vₛₐₘₚₗₑ ÷ TSimplified Formula: PEPCK (nmol/min/ml) = 3215 × ΔA2. 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 × ΔAPrecautionsBefore formal assay, it is essential to perform a pilot test with 2-3 samples expected to have significant differences in activity... Read More | Product content： Component G665666 200 preps Buffer P1 60ml Buffer P2 60ml Buffer E3 60ml Buffer PW (concentrate) 25ml Buffer EB 30ml RNase A (10 mg/ml) 600 µl Spin Columns DM 200 with Collection Tubes 200Product Introduction:This reagent kit is suitable for extracting 1-5 ml of Product content： Component G665666 200 preps Buffer P1 60ml Buffer P2 60ml Buffer E3 60ml Buffer PW (concentrate) 25ml Buffer EB 30ml RNase A (10 mg/ml) 600 µl Spin Columns DM 200 with Collection Tubes 200Product Introduction:This reagent kit is suitable for extracting 1-5 ml of bacterial solution. On the basis of alkaline lysis of cells, it efficiently and specifically binds plasmid DNA through a new silicon-based membrane. Each adsorption column can adsorb up to 40% µ The plasmid DNA of g is effectively removed with a special buffer system to effectively remove impurities such as proteins. The yield and purity of plasmids obtained from this kit are high, and the quality is stable. It is suitable for downstream experiments such as cell transfection, DNA sequencing, PCR, PCR based mutations, in vitro transcription, transformed bacteria, and endonuclease digestion.Self prepared reagents: anhydrous ethanol, isopropanol.Preparation and important precautions before the experiment:1. All components can be stably stored for 1 year in a dry, room temperature (15-30 ℃) environment. The adsorption column can be stored for a longer time at 2-8 ℃. 2.Buffer P1 with RNase A added can be stably stored for 6 months at 2-8 ℃. Before use, add RNase A to Buffer P1 (add all RNase A provided in the reagent kit), mix well, and store at 2-8 ℃. Before use, it is necessary to leave it at room temperature for a period of time, and then use it after returning to room temperature.3.Before the first use, anhydrous ethanol should be added to the Buffer PW according to the instructions on the reagent bottle label.4. Before use, please check if there is any crystallization or precipitation in Buffer P2 and Buffer E3. If there is any crystallization or precipitation, you can take a water bath at 37 ℃ for a few minutes to restore clarity.5. Note that Buffer P2 and Buffer E3 contain irritating substances. Please wear gloves when operating and immediately cover the lid after use.6.The amount and purity of plasmid extraction are related to factors such as bacterial culture concentration, strain type, plasmid size, and plasmid copy number.7. The maximum volume of Spin Columns DM is 750 µ l. If the sample volume is greater than 750 µ L can be added in batches.Operation steps：1. Take 1-5 ml of overnight cultured bacterial solution and add it to a centrifuge tube (provided). Centrifuge at 13000 rpm (~16200 × g) for 1 minute to collect bacteria, and try to discard all the supernatant as much as possible.2. Add 200 to the centrifuge tube containing bacterial sediment µ Buffer P1 (please check if RNase A has been added first), mix thoroughly with a pipette or vortex oscillator, and suspend bacterial precipitation.Attention: If the bacterial blocks are not thoroughly mixed, it will affect the cracking effect, resulting in low extraction amount and purity.3. Add 200 to the centrifuge tube µ Buffer P2, gently invert and mix 8-10 times to fully lyse the bacterial cells. At this point, the solution should become clear and viscous.Attention: Mix gently and do not shake vigorously to avoid interrupting genomic DNA and mixing genomic DNA fragments in the extracted plasmid. If the solution does not become clear, it indicates that the bacterial count may be too large and the lysis may not be complete. The bacterial count should be reduced or the dosage of P1, P2, E3, and isopropanol should be increased proportionally.4. Add 200 to the centrifuge tube µ Buffer E3, immediately invert and mix 8-10 times, at which point white flocculent precipitates appear. Centrifuge at 13000 rpm for 5 minutes.Attention: After adding Buffer E3, it should be mixed evenly immediately to avoid local precipitation.5. Add 260 to the spin columns DM that have been loaded into the collection tube µ After adding isopropanol, immediately add the supernatant collected in step 4 and mix it upside down.Attention: After adding isopropanol, immediately add the supernatant and mix well to avoid isopropanol dripping into the collection tube after being left for a long time. The maximum volume of the adsorption column is 750 µ l. If the sample volume is greater than 750 µ l. Isopropanol and the supernatant can be collected in a centrifuge tube (provided by oneself), mixed well, and passed through the column in batches.6.13000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7. Add 400 to the adsorption column µ L Buffer PW (please check if anhydrous ethanol has been added first), centrifuge at 13000 rpm for 1 minute, and discard the waste liquid in the collection tube.8. Place the adsorption column in a new collection tube and add 50-100 to the middle of the adsorption membrane µ Centrifuge at 13000 rpm for 1 minute using buffer EB and collect the plasmid solution into a centrifuge tube- Store the plasmid at 20 ℃.Note: 1) To increase the efficiency of plasmid recovery, the obtained solution can be added back to the adsorption column, left at room temperature for 2-5 minutes, centrifuged at 13000 rpm for 2 minutes, and collected into a centrifuge tube.2) When the plasmid copy number is low or>10 kb, preheating the buffer EB in a water bath at 65-70 ℃ can increase the extraction efficiency... Read More | Product introduction：Reporter gene detection is an important tool for analyzing the interaction between potential cis elements (such as promoters, enhancers and silencers) and trans acting factors in the flanking region of structural genes in the field of modern molecular biology. Firefly Product introduction：Reporter gene detection is an important tool for analyzing the interaction between potential cis elements (such as promoters, enhancers and silencers) and trans acting factors in the flanking region of structural genes in the field of modern molecular biology. Firefly luciferase is widely used in gene regulation and drug screening. Firefly luciferase is a protein with a molecular weight of about 61 KD. In the presence of ATP, magnesium ions and oxygen, it can catalyze the production of oxyluciferin from luciferin. In the process of luciferin oxidation, it will produce a light signal. The optical signal of this kit is a kind of instantaneous light, which needs to be detected immediately after adding the working solution. The half-life of optical signal is about 5 min.Instruction：1.Working fluid configuration ( 1 ) Restore all components to room temperature. ( 2 ) The component B ( stock solution ) was fully diluted with component A to prepare a 0.2 mg / mL firefly luciferase working solution, which was vortexed and shaken to ensure full mixing. Note : The firefly luciferase working solution cannot be repeatedly frozen and thawed. If the dosage of a single experiment is small, it is recommended to subpackage according to a single dosage. At room temperature, the activity decreased by about 10 % after the working solution was configured for 3 h, and the activity decreased by about 25 % after 5 h. 2.chemiluminescence value detection ( 1 ) The cell culture plate was taken out from the incubator and incubated at room temperature for 20 min to restore it to room temperature ( 22-25 ° C ). ( 2 ) Add the same volume of firefly luciferase working solution with the medium to the culture plate and mix well. ( 3 ) Incubation at room temperature for 5 min. Note : The incubation time can be adjusted according to cell type and cell number. ( 4 ) The values were read by multifunctional microplate reader or chemiluminescence instrument ( instrument parameters : the determination time was 10 s, the determination interval was 2 s ).Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. the strongest wavelength of bioluminescence catalyzed by firefly luciferase is 560 nm. 3. to prevent interference between holes, it is recommended to use white opaque orifice plate.Recommendation：Component B is recommended to use sterile water in advance to configure 2 mg / mL storage solution, A component and B component configured as storage solution, and small batch packaging according to the experimental requirements. The detection working fluid is recommended to be used now to avoid repeated freezing and thawing. Component：One-Step Firefly Luciferase Assay Buffer；D-Luciferin Scope of application：Mainly used for ADCC detection... Read More | Product content： Component O66550510 preps O665505 50 preps Blocking Buffer 100 ml 500 ml Antibody Pretreat Solution（ HRP/Mouse ） 1 ml 5 × 1 ml Dilution Buffer 100 ml 500 ml Wash Buffer（ 10× ) 100 ml 500 mlProduct Introduction:The one-step rapid WB assay kit (Product content： Component O66550510 preps O665505 50 preps Blocking Buffer 100 ml 500 ml Antibody Pretreat Solution（ HRP/Mouse ） 1 ml 5 × 1 ml Dilution Buffer 100 ml 500 ml Wash Buffer（ 10× ) 100 ml 500 mlProduct Introduction:The one-step rapid WB assay kit (mouse) is the latest Western Blot detection kit developed by Kangwei Century, which can obtain high-quality Western Blot results in about 1 hour. It is easy to operate, has high detection sensitivity, low background, does not require the addition of secondary antibodies, and has strong system stability. The conventional Western Blot indirect detection process (blocking, primary antibody binding, and secondary antibody binding) requires a long time, a complex experimental process, and requires multi-step condition optimization. After transferring the protein on the gel to the carrier membrane, incubate it with the blocking solution in the reagent kit for 5 minutes, and then incubate the carrier membrane with the primary antibody treated with antibody reaction solution. After washing three times (5 minutes each time), luminescence or colorimetric detection can be performed. This reagent kit is designed for use in experimental systems where the target protein primary antibody is derived from mice.Notes:1. The customer prepares their own mouse source primary antibody.2. Before using Blocking Buffer blocking solution, Antibody Pretreat Solution (HRP/Mouse) antibody reaction solution (mouse), and Wash Buffer (10 x) rinse solution, please mix thoroughly.3. If there is precipitation in the rinsing solution when stored at 2-8 ℃, please restore it to room temperature, dissolve the precipitation, and use it normally. The 1x rinsing solution can be stored at room temperature for one month.4. It is recommended to stain the membrane with reagents such as spring red after the transfer is completed, and cut off any excess parts on the membrane to increase the efficiency of the reagents.5. The optimal dilution amount for primary antibody and antibody reaction solution HRP (mouse) needs to be determined through preliminary experiments.6. The antibody reaction solution HRP (mouse), antibody dilution solution, and antibody dosage can be increased or decreased proportionally according to the size of the membrane.7. The antibody dilution solution containing the first antibody can be recycled and reused once. It is recommended not to reuse antibodies with poor specificity and affinity. If the recovered antibody is used within 1-2 days and stored at 2-8 ℃ for long-term storage, please freeze it at -20 ℃ to avoid repeated freeze-thaw cycles.8. If there is a high background, please adjust the amount of antibodies and increase the number of times the film is washed.9. All reagents in the reagent kit should be stored at 2-8 ℃ to avoid freezing and thawing.Operation steps:This product is suitable for the sealing and antibody incubation steps after membrane transfer, taking a 5 cm x 8 cm membrane as an example:1. Preparation of rinsing solution: Dilute 10 ml of Wash Buffer (10 x) with distilled water to 100 ml, which is 1 x Wash Buffer. Set aside. Use 8-10 ml for each film wash.2. Sealing: After the membrane transfer is completed, immerse the membrane in 10 ml Blocking Buffer and seal at room temperature for 5 minutes.3. Rinse: Pour off the sealing solution, add 8-10 ml of 1 x Wash Buffer, and rinse at a high speed on a shaker for 1 minute.4. Prepare antibody incubation solution while washing the membrane: Take Antibody Pretreat Solution (HRP/Mouse) 100 µ Add mouse derived primary antibody 3-10 into the centrifuge tube µ g. Suck and beat the gun head until thoroughly mixed, and incubate at room temperature for 5 minutes. Add to 10 ml Dilution Buffer and mix well. Note: 1) The dosage of primary antibody can also be adjusted according to the dilution of the antibody. Taking the final dilution of antibodies at 1:1000 as an example, take 100 µ Add HRP (mouse) antibody reaction solution into the EP tube and add 10 µ Add the first antibody to 10 ml of antibody diluent, mix well, and incubate at room temperature for 5 minutes. 2) If the membrane area is small, the amount of antibodies, reaction solution, and diluent can be reduced proportionally.5. After completing step 3, pour out the rinsing solution and add the antibody incubation solution mixed with primary antibody, Antibody Pretreat Solution (HRP/Mouse), and Dilution Buffer to the membrane (ensuring that the incubation solution completely submerges the surface of the membrane). Incubate at room temperature on a shaker at around 60 rpm for 40 minutes.6. Discard (recover) the antibody incubation solution and rinse 3-5 times with the prepared 1 x Wash Buffer, each time for 3 minutes. 7. Conduct subsequent testing. It is recommended to use ECL or DAB methods for testing.Application examples:Example 1 Antigen is 293T cell lysateA: Normal WB control: beta actin mouse monoclonal antibody (CW0096) 5 µ Incubate at room temperature for 40 minutes, wash the film and dilute the secondary antibody sheep anti mouse HRP (CW0102) 1:10000. Incubate at room temperature for 40 minutes and expose ECL (CW0049).B: One step method WB: beta actin mouse monoclonal antibody (CW0096) 5 µ Incubate at room temperature for 40 minutes and expose ECL (CW0049).Example 2 Antigen is E. coli multi label protein lysateC: Normal WB control: GST mouse monoclonal antibody (CW0084) 2.5 µ Incubate at room temperature for 40 minutes, wash the film and dilute the secondary antibody sheep anti mouse HRP (CW0102) 1:10000. Incubate at room temperature for 40 minutes and expose ECL (CW0049).D: One step method WB: GST mouse monoclonal antibody (CW0084) was incubated at room temperature with 2.5ug for 40 minutes, and ECL (CW0049) was exposed... Read More | Inquire |