Pyruvate Kinase Assay Kit (Amplex Red), IC-6573
Product Introduction
This product is a sensitive and specific fluorescence-based enzymatic assay kit for the quantitative measurement of pyruvate kinase (PK) activity in biological samples. Pyruvate kinase catalyzes the final rate-limiting step of glycolysis, converting phosphoenolpyruvate (PEP) and ADP to pyruvate and ATP, and plays a central role in regulating glycolytic flux and cellular energy metabolism. PK exists as four tissue-specific isozymes (PKL, PKR, PKM1, PKM2), with the M2 isoform (PKM2) frequently overexpressed in cancer cells and recognized as a key driver of aerobic glycolysis (the Warburg effect) and tumor metabolism. This kit employs a coupled enzymatic reaction system in which pyruvate kinase first converts PEP and ADP to pyruvate and ATP, after which pyruvate oxidase converts pyruvate to acetyl phosphate and H₂O₂. In the presence of horseradish peroxidase (HRP), H₂O₂ reacts with Amplex Red reagent to generate the highly fluorescent product resorufin. The fluorescence signal is directly proportional to pyruvate kinase activity in the sample. The assay is compatible with a wide range of biological samples including cell lysates, tissue homogenates, and purified enzyme preparations.
Product Features
1. Sensitive Fluorometric Detection: Amplex Red-based assay enables quantification of pyruvate kinase activity with excellent signal-to-noise ratios.
2. Coupled Enzymatic System: Specific enzymatic cascade provides reliable measurement of PK activity with minimal interference from other glycolytic enzymes.
3. Versatile Sample Compatibility: Suitable for cell lysates, tissue homogenates, and purified enzyme preparations.
4. Convenient 96-Well Format: Compatible with high-throughput workflows and standard fluorescence microplate readers.
5. Complete Kit Format: Includes all necessary enzymes, cofactors, Amplex Red reagent, and pyruvate kinase standard for a complete assay workflow.
Specifications
Size: 100 assays
Detection Method: Fluorescence (Ex/Em: 530-560 nm / 570-595 nm)
Format: 96-well plate
Sample Types: Cell lysates, tissue homogenates, purified enzyme preparations
Storage and Stability
Storage Conditions: Store kit components at -20¡ãC, protected from light. The Amplex Red reagent is particularly light-sensitive and must be stored protected from light.
Shelf Life: The product is stable for 12 months from the date of manufacture when stored as directed. Avoid repeated freeze-thaw cycles.
Protocol (For Reference Only)
Important: Protect Amplex Red reagent and working solutions from light at all times. Prepare all working solutions freshly before use. Maintain samples on ice during preparation to preserve enzyme activity.
1. Sample Preparation: Prepare cell or tissue lysates in appropriate lysis buffer without phosphatase or kinase inhibitors that may interfere with pyruvate kinase activity. Centrifuge lysates at 10,000 x g for 10 minutes at 4¡ãC and collect the supernatant. Determine protein concentration by BCA or Bradford assay for normalization.
2. Standard Curve Preparation: Prepare a serial dilution of the provided pyruvate kinase standard in assay buffer to generate a standard curve. Include a blank well containing assay buffer only.
3. Reaction Setup: Add the appropriate volume of each standard, diluted sample, and blank to designated wells of a black 96-well microplate. Prepare Amplex Red Working Solution by mixing the provided components as directed in the detailed protocol, and add to each well.
4. Incubation: Incubate the plate at 37¡ãC for 30 minutes protected from light. Monitor fluorescence development periodically.
5. Detection and Analysis: Measure fluorescence using a microplate reader at excitation 530-560 nm and emission 570-595 nm. Subtract blank values, generate a standard curve, and calculate pyruvate kinase activity in unknown samples by interpolation.
Precautions
1. Amplex Red reagent is highly light-sensitive; prepare working solutions immediately before use and protect from light during incubation.
2. Maintain samples on ice and process promptly to preserve pyruvate kinase enzymatic activity.
3. Certain reducing agents and detergents may interfere with the HRP-catalyzed reaction; avoid contamination of reagents and samples.
4. For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: What isozymes of pyruvate kinase does this kit detect?
A1: The assay measures total pyruvate kinase activity independent of isozyme type, detecting all four tissue-specific isoforms (PKL, PKR, PKM1, PKM2). For isoform-specific measurement, immunoprecipitation or isoform-specific antibodies should be used prior to the assay.
Q2: Can I use this kit for tissue homogenates?
A2: Yes. The kit is compatible with tissue homogenates. Homogenize tissue in appropriate lysis buffer, centrifuge to remove debris, and use the supernatant for analysis. Normalize activity to tissue weight or protein concentration.
Disclaimer
1. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
2. 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.
3. Wear appropriate protective clothing and gloves when handling this product.
Ordering Information
Catalog Number: IC-6573
Product Name: Pyruvate Kinase Assay Kit (Amplex Red)
Size: 100 assays
Price: CNY ¥1510.00 / USD $151.00 / EUR €181.20 / JPY ¥27180.00
