L-Lactate Assay Kit, IC-6571
Product Introduction
This product is a colorimetric assay kit for the quantitative measurement of L-lactate in biological samples. Lactate is a key metabolite produced during anaerobic glycolysis and serves as an important indicator of cellular metabolic status, tissue oxygenation, and mitochondrial function. Elevated lactate levels are associated with hypoxia, ischemia, sepsis, and tumor metabolism (the Warburg effect). This kit employs a coupled enzymatic reaction system in which lactate dehydrogenase (LDH) specifically catalyzes the oxidation of L-lactate to pyruvate, with the concurrent reduction of NAD⁺ to NADH. The NADH generated then reduces nitrotetrazolium blue chloride (NBT) via the electron carrier N-methylphenazinium methyl sulfate (PMS) to produce a purple-colored formazan product. The absorbance of this product at 530 nm is directly proportional to the lactate concentration in the sample. This colorimetric method provides a simple, sensitive, and specific measurement of L-lactate without requiring fluorescence detection equipment, making it accessible to a broad range of laboratory settings.
Product Features
1. Colorimetric Detection: Absorbance measurement at 530 nm enables quantification using standard spectrophotometers or microplate readers without specialized fluorescence equipment.
2. Specific Enzymatic Detection: LDH-catalyzed oxidation provides high specificity for L-lactate with minimal cross-reactivity toward D-lactate or other metabolites.
3. High Sensitivity: Detection limit of 0.10 mmol/L enables accurate measurement of low lactate concentrations in biological samples.
4. Wide Linear Range: Linear detection range of 0.12-7.0 mmol/L accommodates diverse sample types and experimental conditions without extensive pre-dilution.
5. Complete Kit Format: Includes all necessary enzymes, cofactors, chromogenic substrate, and L-lactate standard for a complete assay workflow.
Specifications
Size: 100 assays
Detection Method: Colorimetric (absorbance at 530 nm)
Assay Principle: LDH-catalyzed oxidation of lactate coupled with PMS-mediated NBT reduction
Detection Indicator: LA (Lactic Acid) colorimetric method
Sensitivity: 0.10 mmol/L
Detection Range: 0.12-7.0 mmol/L
Format: 96-well plate compatible
Sample Types: Cell culture supernatants, serum, plasma, tissue homogenates
Storage and Stability
Storage Conditions: Store kit components at -20¡ãC, protected from light. The NBT 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. Reconstituted reagents should be used immediately or stored at -20¡ãC for up to 1 week.
Protocol (For Reference Only)
Important: Protect NBT and PMS reagents from light at all times. Prepare all working solutions freshly before use. Use ultrapure water and clean glassware to prevent lactate contamination.
1. Sample Preparation: For cell culture supernatants, centrifuge at 1,000 x g for 5 minutes to remove cells and debris. For serum or plasma, dilute 1:10 to 1:100 in assay buffer to bring lactate concentrations within the linear range. For tissue homogenates, homogenize tissue in cold PBS, centrifuge at 10,000 x g for 10 minutes at 4¡ãC, collect the supernatant, and dilute as needed. Lactate is rapidly metabolized; process samples promptly and store at -80¡ãC if analysis is delayed.
2. Standard Curve Preparation: Prepare a serial dilution of the provided L-lactate standard in assay buffer to generate a standard curve covering the range of 0-7.0 mmol/L. 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 clear 96-well microplate. Prepare the LDH-NBT-PMS 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 color development periodically; the reaction typically reaches completion within 30 minutes.
5. Detection and Analysis: Measure absorbance at 530 nm using a microplate reader or spectrophotometer. Subtract the blank value from all readings. Generate a standard curve by plotting absorbance versus standard concentration, and calculate L-lactate concentrations in unknown samples by interpolation, correcting for any dilution factors applied during sample preparation.
Precautions
1. Protect NBT and PMS reagents from light throughout the procedure; light exposure reduces assay sensitivity and reproducibility.
2. Lactate is a common environmental contaminant found in sweat; wear gloves and use clean glassware or plasticware throughout the procedure.
3. Certain reducing agents (e.g., ascorbic acid, ¦Â-mercaptoethanol) may interfere with the NBT reduction reaction; avoid contamination of reagents and samples.
4. L-lactate is rapidly produced and consumed by living cells; process cell culture samples promptly and snap-freeze for storage if analysis cannot be performed immediately.
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: Lactate dehydrogenase (LDH) catalyzes the oxidation of lactate to pyruvate, reducing NAD⁺ to NADH. The NADH then reduces NBT to a purple formazan product via the electron carrier PMS. The absorbance at 530 nm is directly proportional to the lactate concentration.
Q2: Does this kit detect D-lactate?
A2: No. The LDH used in this kit is specific for L-lactate, the physiologically relevant isomer in mammalian metabolism. D-lactate, which may be elevated in certain bacterial infections, is not detected.
Q3: How should I dilute cell culture medium samples?
A3: Typical L-lactate concentrations in spent culture medium from rapidly proliferating cells range from 1-10 mmol/L. A dilution factor of 1:50 to 1:500 is generally appropriate. For cells with high glycolytic rates, higher dilutions may be necessary. Perform a preliminary experiment to determine the optimal dilution.
Q4: What is the advantage of this colorimetric method over fluorometric methods?
A4: Colorimetric detection at 530 nm can be performed on standard absorbance microplate readers and spectrophotometers without requiring specialized fluorescence detection equipment, making this kit accessible to a broader range of laboratory settings.
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 and experimental systems.
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 and biological samples.
Ordering Information
Catalog Number: IC-6571
Product Name: Lactate Assay Kit
Size: 100 assays
Price: CNY ¥1950.00 / USD $195.00 / EUR €234.00 / JPY ¥35100.00
2023 Version
