Acetyl-CoA Assay Kit, IC-6561
Product Introduction
Acetyl-coenzyme A (acetyl-CoA) is a central metabolite at the intersection of carbohydrate, lipid, and protein metabolism. It serves as the primary substrate for the tricarboxylic acid (TCA) cycle, the building block for fatty acid and cholesterol biosynthesis, and the acetyl donor for protein acetylation reactions. Acetyl-CoA levels reflect cellular metabolic status and are dysregulated in obesity, diabetes, cancer, and neurodegenerative disorders. This kit employs a coupled enzymatic reaction system for the specific and quantitative measurement of acetyl-CoA in biological samples. The assay principle is based on the sequential reactions catalyzed by malate dehydrogenase (MDH) and citrate synthase (CS). Malate dehydrogenase catalyzes the conversion of malate and NAD⁺ to oxaloacetate and NADH. Citrate synthase then catalyzes the condensation of acetyl-CoA with oxaloacetate to generate citrate and free coenzyme A, releasing the oxaloacetate consumed in the preceding step. These coupled reactions result in a continuous cycling of oxaloacetate, with the net reaction being the acetyl-CoA-dependent reduction of NAD⁺ to NADH. The rate of NADH production, measured as the increase in absorbance at 340 nm, is directly proportional to the acetyl-CoA concentration in the sample.
Product Features
1. Specific Enzymatic Detection: Coupled MDH-CS enzyme system provides high specificity for acetyl-CoA with minimal interference from other acyl-CoA species.
2. Kinetic Absorbance Measurement: Continuous monitoring of NADH production at 340 nm enables accurate quantification using standard spectrophotometers or microplate readers.
3. Broad Sample Compatibility: Validated for use with cell lysates, tissue homogenates, and other biological samples.
4. Complete Kit Format: Includes all necessary enzymes, cofactors, substrates, and acetyl-CoA standard for a complete assay workflow.
5. Flexible Throughput: Available in 24, 48, and 96 reaction sizes to accommodate different experimental scales.
Specifications
Size: Available in 24 reactions, 48 reactions, and 96 reactions
Detection Method: Absorbance kinetics at 340 nm
Assay Principle: Coupled malate dehydrogenase and citrate synthase enzymatic cycling reaction
Detection Indicator: NADH production rate (¦¤A340/min)
Sample Types: Cell lysates, tissue homogenates, biological fluids
Storage and Stability
Storage Conditions: Store kit components at -20¡ãC, protected from light and moisture.
Shelf Life: The product is stable for 12 months from the date of manufacture when stored as directed. Avoid repeated freeze-thaw cycles of reconstituted reagents.
Protocol (For Reference Only)
Important: Prepare all working solutions freshly before use. Maintain samples on ice during preparation to preserve acetyl-CoA stability. Acetyl-CoA is labile and susceptible to hydrolysis; process samples promptly.
1. Sample Preparation: For cultured cells, harvest 2-5¡Á10⁶ cells, wash once with cold PBS, and homogenize in ice-cold assay buffer or acetyl-CoA extraction buffer. Centrifuge at 10,000 x g for 10 minutes at 4¡ãC and collect the supernatant. For tissue samples, homogenize 20-50 mg of tissue in extraction buffer, centrifuge as above, and collect the supernatant. Deproteinization using perchloric acid or ultrafiltration may be required for samples with high protein content.
2. Standard Curve Preparation: Prepare a serial dilution of the provided acetyl-CoA standard in assay buffer to generate a standard curve typically covering 0.1-10 nmol per well. Include a blank well containing assay buffer only.
3. Reaction Setup: Add the appropriate volume of each standard, sample, and blank to designated wells of a clear 96-well plate suitable for UV absorbance measurement. Prepare the MDH-CS Working Solution containing malate dehydrogenase, citrate synthase, malate, and NAD⁺ as directed in the detailed protocol. Add the working solution to each well to initiate the reaction.
4. Kinetic Measurement: Immediately place the plate in a microplate reader pre-warmed to 25¡ãC or 37¡ãC. Measure the absorbance at 340 nm continuously for 10-30 minutes, recording readings at 30-second or 1-minute intervals. The absorbance should increase linearly over time.
5. Data Analysis: Calculate the reaction rate (¦¤A340/min) for each standard and sample by linear regression of the initial linear portion of the kinetic curve. Subtract the blank rate from all readings. Generate a standard curve by plotting reaction rate versus acetyl-CoA concentration, and calculate acetyl-CoA concentrations in unknown samples by interpolation. Normalize to protein concentration or tissue weight.
Precautions
1. Acetyl-CoA is labile in aqueous solution; maintain samples at 4¡ãC and process promptly to minimize hydrolysis.
2. The assay requires UV-transparent 96-well plates for absorbance measurement at 340 nm; standard polystyrene plates absorb UV light and are unsuitable.
3. NADH production rate should be linear during the measurement period; non-linear kinetics may indicate interfering substances or enzyme inactivation.
4. Some biological samples may contain NADH-consuming enzymes; sample deproteinization or appropriate controls are recommended.
5. For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: What is the detection principle of this kit?
A1: The assay uses a coupled enzymatic cycling reaction. Malate dehydrogenase converts malate and NAD⁺ to oxaloacetate and NADH. Citrate synthase then condenses acetyl-CoA with oxaloacetate to generate citrate, regenerating oxaloacetate for the next cycle. The rate of NADH production (¦¤A340/min) is directly proportional to acetyl-CoA concentration.
Q2: Why is a kinetic measurement used rather than an endpoint measurement?
A2: Kinetic measurement provides greater accuracy by monitoring the reaction rate over time rather than a single endpoint reading. This minimizes interference from background absorbance, corrects for sample-specific variations, and ensures that measurements are taken within the linear range of the assay.
Q3: How should I prepare tissue samples for this assay?
A3: Rapidly homogenize fresh or frozen tissue in ice-cold extraction buffer. Centrifuge at 10,000 x g for 10 minutes at 4¡ãC to remove debris. For tissues with high protein content, deproteinization using perchloric acid precipitation or ultrafiltration through a 10 kDa cutoff filter is recommended to remove interfering enzymes and proteins.
Disclaimer
1. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
2. Due to the variable nature of biological samples, optimization of sample preparation and extraction conditions is recommended for specific sample types.
3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages, including loss of samples or data.
4. Wear appropriate protective clothing and gloves when handling this product.
Ordering Information
Catalog Number: IC-6561
Product Name: Acetyl-CoA Assay Kit
Size: 24 reactions / 48 reactions / 96 reactions
Price:
24 reactions: CNY ¥1520.00 / USD $152.00 / EUR €182.40 / JPY ¥27360.00
48 reactions: CNY ¥2810.00 / USD $281.00 / EUR €337.20 / JPY ¥50580.00
96 reactions: CNY ¥3890.00 / USD $389.00 / EUR €466.80 / JPY ¥70020.00
2023 Version
