| Description | Acetate Kinase (ACK) is primarily found in microorganisms. It catalyzes the conversion of acetate and ATP to acetyl phosphate and ADP, serving as a key enzyme in bacterial carbon and energy metabolism, and plays a central role particularly in the methanogenesis metabolism of archaea.Assay Acetate Kinase (ACK) is primarily found in microorganisms. It catalyzes the conversion of acetate and ATP to acetyl phosphate and ADP, serving as a key enzyme in bacterial carbon and energy metabolism, and plays a central role particularly in the methanogenesis metabolism of archaea.Assay PrincipleACK catalyzes the synthesis of Acetyl Phosphate and ADP from Sodium Acetate and ATP. Pyruvate Kinase then catalyzes the conversion of ADP and Phosphoenolpyruvate (PEP) to ATP and Pyruvate. Subsequently, Lactate Dehydrogenase catalyzes the reduction of Pyruvate by NADH to produce Lactate and NAD⁺. The rate of oxidation of NADH to NAD⁺, measured by the decrease in absorbance at 340 nm, reflects ACK activity.Component50TStorageExtraction Buffer80 mL2-8℃Reagent 11EA-20℃Reagent 24EA-20℃Reagent 32EA-20℃Reagent 42EA-20℃Reagent PreparationReagent 1: Before use, centrifuge the vial to bring the powder to the bottom. Add 8.4 mL of distilled water to dissolve. Aliquot and store unused portions at -20°C.Reagent 2: Before use, centrifuge each vial to bring the powder to the bottom. Add 0.6 mL of distilled water to each vial to dissolve. Aliquot and store unused portions at -20°C. Avoid repeated freeze-thaw cycles. Use within 3 days.Reagent 3: Before use, centrifuge each vial to bring the powder to the bottom. Add 0.6 mL of distilled water to each vial to dissolve thoroughly. Can be aliquoted and frozen. Avoid repeated freeze-thaw cycles.Reagent 4: Before use, centrifuge each vial to bring the powder to the bottom. Add 0.6 mL of distilled water to each vial to dissolve thoroughly. Can be aliquoted and frozen. Avoid repeated freeze-thaw cycles.Required Materials and Equipment (Not Provided)UV spectrophotometer, 1 mL quartz cuvette (1 cm light path), refrigerated benchtop centrifuge, constant temperature incubator, pipettes, mortar and pestle, ice, and distilled water.Sample Preparation1.Tissue Samples: Weigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and homogenize on ice. Centrifuge the homogenate at 12,000 rpm (approx. 13,000-15,000 g), 4°C for 10 min. Collect the supernatant and keep it on ice for assay.Note: For larger samples, use an Extraction Buffer volume (mL) to tissue mass (g) ratio between 5:1 and 10:1.2.Bacteria/Cell Samples: Collect cells by centrifugation and discard the supernatant. Add 1 mL of Extraction Buffer per 5 million cells/bacteria. Disrupt the cells/bacteria by sonication on ice (20% power or 200W, pulse 3s on/10s off, repeat 30 times). Centrifuge the lysate at 12,000 rpm, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.Note: For larger samples, use an Extraction Buffer volume (mL) to cell count (10⁴ cells) ratio between 1:1000 and 1:5000.Assay Procedure:1.Preheat the UV spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. Zero the instrument with distilled water.2.Pre-warm all reagents at 37°C for 5-15 minutes.3.Optional Master Mix: A Master Mix can be prepared just before use by combining Extraction Buffer, Reagent 1, Reagent 2, Reagent 3, and Reagent 4 in a 400:160:40:20:20 ratio (e.g., for one assay: 400µL + 160µL + 40µL + 20µL + 20µL = 640µL). Pipette 640 µL of this Master Mix per cuvette. Prepare the Master Mix fresh for immediate use.4.Pipette into a 1 mL quartz cuvette (1 cm light path) in the following order:ReagentVolume (µL)Extraction Buffer400Reagent 1160Reagent 240Reagent 320Reagent 420Mix thoroughly and incubate at 37°C for 5 min.Sample60Mix thoroughly immediately after adding the sample. Record the initial absorbance (A₁) at 340 nm at 10 seconds. Record the absorbance again (A₂) after exactly 10 minutes. Calculate ΔA = A₁ - A₂.Notes & Troubleshooting:1.If ΔA is close to zero, consider extending the reaction time (e.g., to 20 min) to read A₂. Use the new reaction time (T) in the calculation. Alternatively, increase the sample volume V₁ (e.g., to 100 µL, decrease Extraction Buffer accordingly) and use the new V₁ and T in the calculation.2.If the initial absorbance A₁ is too high (e.g., >2, common in deeply pigmented plant samples), reduce the sample volume V₁ and use the new V₁ in the calculation. Alternatively, add a small amount of activated charcoal to the sample supernatant, mix, let stand for 5 min, centrifuge at 12,000 rpm, 4°C for 10 min, and use the clarified supernatant for assay.3.If ΔA is greater than 0.6, reduce the reaction time (e.g., to 5 min) and use the new time (T) in the calculation.4.If the decrease is not linear, read the absorbance every 10 seconds and select a linear segment for calculating ΔA. Use the corresponding time interval (T) for the calculation.ACK Activity Calculation:General Parameters:ε (NADH molar extinction coefficient) = 6.22 × 10³ L/mol/cmd (Cuvette light path) = 1 cmV (Total extraction volume) = 1 mLV₁ (Sample volume in reaction) = 0.06 mL (60 µL)V₂ (Total reaction volume) = 0.0007 L (700 µL)T (Reaction time) = 10 min500 (Cell/Bacteria count in ten-thousands: 500 × 10⁴ = 5 million)W (Sample mass, g)Cpr (Sample protein concentration, mg/mL)1. 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:ACK Activity (nmol/min/mg prot) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (Cpr × V₁ / V) ÷ TSimplified Formula: ACK (nmol/min/mg prot) = 187.6 × ΔA ÷ Cpr2. 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 fresh tissue.Calculation:ACK Activity (nmol/min/g fresh weight) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (W × V₁ / V) ÷ TSimplified Formula: ACK (nmol/min/g fresh weight) = 187.6 × ΔA ÷ W3. Based on Bacterial/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/bacteria.Calculation (for 5 million cells in 1 ml extract):ACK Activity (nmol/min/10⁴ cell) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (500 × V₁ / V) ÷ TSimplified Formula: ACK (nmol/min/10⁴ cell) = 0.38 × 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 IntroductionBCIP (5-Bromo-4-chloro-3-indolyl phosphate) 5-bromo-4-chloro-3-indolyl-phosphate + NBT (tetrazolium nitro blue) is the best substrate for alkaline phosphatase (AP) One of the combination. Under the catalysis of alkaline phosphatase, BCIP will be hydrolyzed to produce a highly Product IntroductionBCIP (5-Bromo-4-chloro-3-indolyl phosphate) 5-bromo-4-chloro-3-indolyl-phosphate + NBT (tetrazolium nitro blue) is the best substrate for alkaline phosphatase (AP) One of the combination. Under the catalysis of alkaline phosphatase, BCIP will be hydrolyzed to produce a highly reactive product, which reacts with NBT to form an insoluble dark blue to blue-violet compound. This kit can be used for the enzymatic color development of IHC and Western Blot experiments of the AP system. Under AP catalysis, a dark blue precipitate is produced where AP conjugates are combined on tissue sections or blotting membranes. The location and expression of the target protein can be determined based on the color reaction.Product Components40×BCIP: 1 ml40×NBT: 1 mlBCIP/NBT Buffer: 40 mlPrecautions1. The working fluid should be prepared for immediate use, and the prepared working fluid will be effective within 1 hour.2. The amount of working fluid must be sufficient to ensure complete coverage of the tissue sheet or blotting membrane. To3. In order to obtain the best experimental results, be sure to optimize the experimental conditions.4. NBT is poisonous, please take necessary protective measures when using it.5. This product is only used for scientific research, not for human experiments or human treatment.Instructions1. BCIP/NBT color developing working solution preparation:According to the required amount, mix 40×BCIP, 40×NBT and BCIP/NBT Buffer in a volume ratio of 1:1:38 to form the BCIP/NBT color developing working solution.2. Color rendering:1) Blotting membrane color development: Drop the prepared working solution on the blotting membrane (or pour the blotting membrane into the BCIP/NBT color developing working solution), and incubate for 3-10 minutes at room temperature and dark. After the color development is completed, the film is immersed in water to terminate the reaction.2) Color development of tissue sections or cell slides: Drop an appropriate amount of BCIP/NBT color developing working solution on the tissue sections or cell slides that need color development, and incubate at room temperature for 3-10 minutes in the dark. Observe under the microscope to control the color development time. When the best color development effect is reached, rinse with tap water to stop the color development. After color development, the slices are counter-stained, dehydrated and transparent, and can be stored for a long time after mounting... Read More | Format:2-ComponentEnzyme:Horseradish peroxidase | Product content： O665490Component50 TStorageO665490ABlocking Buffer500 mL2-8℃. Do not freeze.O665490BAntibody Pretreat Solution (HRP/Rabbit)5×1 mL2-8℃. Do not freeze.O665490CDilution Buffer500 mL2-8℃. Do not freeze.O665490DWash Buffer (10×)500 mL2-8℃. Do not Product content： O665490Component50 TStorageO665490ABlocking Buffer500 mL2-8℃. Do not freeze.O665490BAntibody Pretreat Solution (HRP/Rabbit)5×1 mL2-8℃. Do not freeze.O665490CDilution Buffer500 mL2-8℃. Do not freeze.O665490DWash Buffer (10×)500 mL2-8℃. Do not freeze. Product Introduction：The one-step rapid WB assay kit (rabbit) is the latest Western Blot detection kit developed by Kangwei Century, which canObtain high-quality Western Blot results within about 1 hour, with simple operation, high detection sensitivity, low background, and noAdditional secondary antibodies need to be added, with strong system stability. The conventional Western Blot indirect detection process (blocking, primary antibody binding)Combining with secondary antibodies requires a long time, a complex experimental process, and requires multi-step optimization of conditions. The protein on the glue is transferred toAfter coating the carrier membrane, incubate it with the blocking solution in the reagent kit for 5 minutes, and then incubate the carrier with the primary antibody treated with antibody reaction solutionAfter washing the membrane three times (5 minutes each time), it can undergo luminescence or color detection. This reagent kit is designed for target protein oneThe use of an experimental system derived from rabbits.Notes:1. Customers need to prepare their own rabbit source primary antibody.2. Before using Blocking Buffer blocking solution, Antibody Pretreat Solution (HRP/Rabbit) antibody reaction solution (rabbit), 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 (rabbit) needs to be determined through preliminary experiments.6. Antibody reaction solution HRP (rabbit), 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. Antibodies with low specificity and affinity are not recommended for repeated use. 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/Rabbit) 100 µ Add rabbit 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 (rabbit) 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/Rabbit), and Dilution Buffer to the membrane (ensuring that the incubation solution completely submerges the membrane surface). Incubate at room temperature on a shaker at a speed of about 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.Example 1: Antigen 293T cell lysateA: Ordinary WB control: beta actin rabbit antibody (CW0097) 3.3ug incubated at room temperature for 40 minutes, washed with membrane, secondary antibody sheep anti rabbit HRP (CW0103) diluted at 1:10000, room temperature for 40 minutes, ECL (CW0049) exposed Example 2 Antigen is 293T cell lysateC: Ordinary WB control: PAK1, Epitomics rabbit monoclonal antibody 1:1000, incubated at room temperature for 40 minutes, washed with membrane, secondary antibody sheep anti rabbit HRP (CW0103) diluted at 1:10000, room temperature for 40 minutes, ECL (CW0049) exposedD: One step WB: Epitomics rabbit monoclonal antibody was incubated at 1:1000 room temperature for 40 minutes, and ECL (CW0049) was exposed... Read More | Inquire |