¦Á-Ketoglutarate(¦Á-KG)Content Assay Kit, IC-6532
Product Introduction
¦Á-Ketoglutarate (¦Á-KG) is an organic acid that plays an important role in cellular metabolism and serves as a key precursor for the synthesis of multiple amino acids and proteins. It is widely used in the food, pharmaceutical, chemical, and cosmetic industries. ¦Á-Ketoglutarate is a key intermediate metabolite in the tricarboxylic acid (TCA) cycle and is often used as an important indicator for evaluating metabolic disorders. For example, during cuproptosis, copper ion accumulation causes abnormal oligomerization of lipoylated proteins, leading to TCA cycle dysfunction. Therefore, detecting changes in ¦Á-ketoglutarate and pyruvate levels is also an indicator of cuproptosis. This kit is based on a fluorometric or colorimetric method for rapid and sensitive detection of ¦Á-ketoglutarate content in biological fluids such as serum, plasma, and urine, as well as in tissues, cells, and culture supernatants. Detection principle: ¦Á-Ketoglutarate reacts with alanine under the catalysis of alanine aminotransferase to generate pyruvate and glutamate; pyruvate is oxidized by pyruvate oxidase to produce H₂O₂; in the presence of HRP, the fluorescent probe reacts with H₂O₂ to generate a strongly red fluorescent product with visible light absorption. Fluorescence intensity or absorbance is proportional to ¦Á-ketoglutarate content, allowing detection by fluorometric or colorimetric methods. The fluorometric method can detect as low as 2 µM ¦Á-ketoglutarate, with good linearity in the 2-200 µM range; sensitivity is approximately ten times that of the colorimetric method. Typically, 0.5-2 µL of serum or plasma sample is sufficient for fluorometric detection. The kit also provides an ¦Á-ketoglutarate standard solution for constructing a standard curve to calculate ¦Á-ketoglutarate content in samples.
Detection Principle:
¦Á-Ketoglutarate + Alanine ¡ú (Alanine aminotransferase) ¡ú Pyruvate + Glutamate
Pyruvate + O₂ + H₂O ¡ú (Pyruvate oxidase) ¡ú Acetate + CO₂ + H₂O₂
H₂O₂ + Probe ¡ú (HRP) ¡ú Fluorescent product
Product Features
1. High Sensitivity: Fluorometric method detects as low as 2 µM ¦Á-ketoglutarate, approximately ten times more sensitive than the colorimetric method.
2. Dual Detection Modes: Supports both fluorometric and colorimetric detection to accommodate different sample concentrations and instrument availability.
3. Broad Sample Compatibility: Suitable for serum, plasma, urine, tissues, cells, and culture supernatants.
4. Complete Kit Format: Includes assay buffer, fluorescent probe, enzyme solutions, cofactor, and ¦Á-ketoglutarate standard.
Specifications
Size: 100 tests (including standard curve)
Components:
Component 1: Assay Buffer, 20 mL, 1 bottle, store at -20¡ãC
Component 2: Fluorescent Probe, 200 µL, 1 tube, store at -20¡ãC protected from light
Component 3: Enzyme Solution A, 200 µL, 1 tube, store at -20¡ãC
Component 4: Enzyme Solution B, 200 µL, 1 tube, store at -20¡ãC
Component 5: Cofactor, 200 µL, 1 tube, store at -20¡ãC protected from light
Component 6: ¦Á-KG Standard (10 mM), 200 µL, 1 tube, store at -20¡ãC
Detection Method: Fluorometric (Ex 560 nm / Em 590 nm) or colorimetric (A570 nm)
Linear Range: 2-200 µM (fluorometric method)
Storage and Stability
Storage Conditions: Store all components at -20¡ãC as indicated on individual labels. Protect the fluorescent probe and cofactor from light.
Shelf Life: 6 months from the date of manufacture when stored as directed.
Materials Required but Not Provided
1. 96-well plate (black opaque plate recommended for fluorometric method; transparent plate for colorimetric method),
2. Microplate reader (fluorometric method: excitation 560 nm, emission 590 nm; colorimetric method: 570 nm)
3. Assay Buffer, pipettes and tips, centrifuge tubes
4. 37¡ãC light-protected incubation conditions
Protocol (For Reference Only)
1. Preparation of Reaction Working Solution
Preparation ratio (per 80 µL working solution):
Assay Buffer: 72 µL
Fluorescent Probe: 2 µL
Enzyme Solution A: 2 µL
Enzyme Solution B: 2 µL
Cofactor: 2 µL
Total volume: 80 µL
Common preparation volumes:
1 reaction well: prepare according to the 80 µL formula above.
10 reaction wells: Assay Buffer 720 µL + Fluorescent Probe 20 µL + Enzyme Solution A 20 µL + Enzyme Solution B 20 µL + Cofactor 20 µL, total volume 800 µL.
20 reaction wells: Assay Buffer 1440 µL + Fluorescent Probe 40 µL + Enzyme Solution A 40 µL + Enzyme Solution B 40 µL + Cofactor 40 µL, total volume 1600 µL.
50 reaction wells: Assay Buffer 3600 µL + Fluorescent Probe 100 µL + Enzyme Solution A 100 µL + Enzyme Solution B 100 µL + Cofactor 100 µL, total volume 4000 µL.
Operation Notes:
1. Add components in the order of "Assay Buffer ¡ú Fluorescent Probe ¡ú Enzyme Solution A ¡ú Enzyme Solution B ¡ú Cofactor" and mix thoroughly.
2. Store the prepared working solution at 4¡ãC or on ice protected from light. Prepare fresh and use within the same day.
3. Before use, centrifuge Enzyme Solution A and Enzyme Solution B briefly to collect liquid and mix to avoid inaccurate volume due to sedimentation.
4. Background control wells: replace Enzyme Solution A with Assay Buffer, keeping other components the same, to subtract sample-intrinsic background.
5. Blank control wells: contain Assay Buffer only, to subtract reagent and plate background.
2. Standard Curve Construction
2.1 Colorimetric Method Standard Curve
1. Take 10 µL of ¦Á-KG standard (10 mM) and add 90 µL of Assay Buffer, mix to prepare 1000 µM ¦Á-KG standard solution.
2. Take 0, 0.4, 1, 2, 4, 10, and 20 µL of the 1000 µM standard solution and add to standard wells of a 96-well plate.
3. Adjust to 20 µL with Assay Buffer to prepare standard solutions at concentrations of 0, 20, 50, 100, 200, 500, and 1000 µM.
2.2 Fluorometric Method Standard Curve
1. Take 2 µL of ¦Á-KG standard (10 mM) and add 98 µL of Assay Buffer, mix to prepare 200 µM ¦Á-KG standard solution.
2. Take 0, 0.5, 1, 2, 5, 10, and 20 µL of the 200 µM standard solution and add to standard wells of a 96-well plate.
3. Adjust to 20 µL with Assay Buffer to prepare standard solutions at concentrations of 0, 5, 10, 20, 50, 100, and 200 µM.
Note: Standard curves should be prepared fresh for each experiment, with at least duplicate wells.
3. Sample Preparation and Loading
1. Add 1-20 µL of sample or diluted sample to sample wells of a 96-well plate.
2. Adjust to 20 µL with Assay Buffer.
3. Also set up:
Blank control wells: contain Assay Buffer only, to subtract reagent background;
Background control wells: reaction working solution without Enzyme Solution A, to subtract sample-intrinsic background (recommended).
4. Optimization of sample dilution factor by pilot experiments is recommended to ensure detection values fall within the standard curve range. For fluorometric method, 0.5-2 µL of serum/plasma sample is typically sufficient; optimize according to sample type.
4. Reaction
1. Add 80 µL of reaction working solution to each well and mix gently.
2. Incubate at 37¡ãC protected from light for 30 minutes.
3. If absorbance or fluorescence is low, the reaction time may be extended to 45 or 60 minutes. Standards and samples should be extended in parallel to ensure consistent conditions.
5. Detection
Colorimetric method: Measure absorbance at A570 using a microplate reader.
Fluorometric method: Measure fluorescence at excitation 560 nm and emission 590 nm.
6. Calculation
1. Plot a standard curve with standard concentration as the x-axis and blank/background-subtracted absorbance or fluorescence as the y-axis.
2. Calculate the ¦Á-ketoglutarate concentration A (µM) in the sample well from the standard curve.
3. Calculate the ¦Á-ketoglutarate concentration in the sample as follows:
C (µM) = A ¡Á n
Where:
C: ¦Á-ketoglutarate concentration in the sample (µM);
A: ¦Á-ketoglutarate concentration in the detection well determined from the standard curve (µM);
n: total sample dilution factor.
For tissue or cell homogenates, further convert to content per mg protein or per gram tissue based on the homogenization ratio; for culture supernatants or body fluids, calculate directly by dilution factor.
Precautions
1. This kit is for research use only and not for clinical diagnosis.
2. The kit has a shelf life of 6 months; store according to label conditions. Component 2 (fluorescent probe) and Component 5 (cofactor) must be protected from light.
3. Prepare reaction working solution fresh and use within the same day; store at 4¡ãC or on ice protected from light after preparation.
4. Before use, centrifuge Enzyme Solution A and Enzyme Solution B briefly to collect liquid and mix to avoid inaccurate volume due to sedimentation.
5. For fluorometric method, a black 96-well plate is recommended to reduce well-to-well fluorescence interference; for colorimetric method, a transparent 96-well plate may be used.
6. Avoid repeated freeze-thaw cycles of samples; for serum, plasma, and urine samples, centrifuge to remove precipitate before detection.
7. Samples containing strong reducing agents, high concentrations of detergents, or obvious pigments may interfere with detection; evaluate dilution factors or set background controls in pilot experiments.
8. Standards and samples should be tested on the same plate under the same reaction conditions; standards should be set up in duplicate.
9. If detection values exceed the standard curve range, dilute the sample and re-test.
10. Reaction time may be appropriately extended based on signal strength, but standards and samples must be processed in parallel.
FAQ (Simplified)
Q1: How do I choose between fluorometric and colorimetric methods?
A1: The fluorometric method is more sensitive and suitable for low-concentration samples (e.g., small volumes of serum/plasma, culture supernatants); the colorimetric method is simpler and suitable for higher-concentration samples or laboratories with limited fluorescence equipment. The standard curve ranges differ between the two methods; prepare standards according to the corresponding method.
Q2: What is the difference between blank control and background control?
A2: Blank control wells contain Assay Buffer only and are used to subtract reagent and plate background; background control wells replace Enzyme Solution A with Assay Buffer and are used to subtract sample-intrinsic background. Both are recommended, especially for complex samples.
Q3: What should I do if the standard curve linearity is poor?
A3: Check whether standards have undergone repeated freeze-thaw cycles, whether loading volumes are accurate, whether Assay Buffer was adjusted to 20 µL, and whether reaction times are consistent; standards should be prepared fresh and set up in duplicate.
Q4: What should I do if sample signal is low?
A4: Appropriately extend the 37¡ãC light-protected reaction time to 45 or 60 minutes; also confirm whether the sample is over-diluted, whether enzyme solutions were mixed, and whether the fluorescent probe and cofactor were protected from light.
Q5: What should I do if sample concentration exceeds the standard curve range?
A5: Dilute the sample appropriately with Assay Buffer and re-test, accounting for the dilution factor n in the calculation.
Disclaimer
1. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
2. Due to the variable nature of biological research, optimization of sample preparation and dilution 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.
4. Wear appropriate protective clothing and gloves when handling this product.
Ordering Information
Catalog Number: IC-6532
Product Name: ¦Á-Ketoglutarate Fluorometric Assay Kit
Size: 100 tests (including standard curve)
Suggested Retail Price: CNY ¥4550.00 / USD $455.00 / EUR €546.00 / JPY ¥81900.00
Note: The specific price is subject to the transaction price in the offline contract. This document is provided for technical reference only and does not constitute any form of sales advertisement or invitation to offer. For product details and purchase inquiries, please contact local sales representatives.
