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You are here:Home » Kits and Assays » Kits for Molecular Biology, BioAssay™ » L-Lactate Assay Kit, BioAssay™, Colorimetric/Fluorometric

L-Lactate Assay Kit, BioAssay™, Colorimetric/Fluorometric

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Specifications

Lactate is generated by lactate dehydrogenase (LDH) under hypoxic or anaerobic conditions. Monitoring lactate levels is, therefore, a good indicator of the balance between tissue oxygen demand and utilization and is useful when studying cellular and animal physiology.
Catalog #L1011-04B
Simple, direct and automation-ready procedures for measuring lactate concentration are very desirable. Lactate assay kit is based on lactate dehydrogenase catalyzed oxidation of lactate, in which the formed NADH reduces a probe into a highly fluorescent product. The fluorescence intensity of this product, measured at lex/em=530/585 nm, is proportional to the lactate concentration in the sample.
ApplicationsDirect Assays: L-lactate in serum, plasma, urine, cell media samples and other biological samples.
Key FeaturesSensitive and accurate. Detection limit of 1uM and linearity up to 50uM L-lactate in 96-well plate assay.
Convenient. The procedure involves adding a single working reagent, and reading the fluorescence after 60 min. Room temperature assay.
High-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.
Kit ContentsAssay Buffer: 10ml Enzyme A: 120ul
NAD Solution: 1ml Enzyme B: 120ul
Probe: 750ul Standard: 1ml
Storage conditions. Store all reagents at -20°C. Shelf life: 6 months after receipt.
Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.
Sample Preparation And ConsiderationsThe following substances interfere and should be avoided in sample preparation: EDTA (>0.5mM), ascorbic acid, SDS (>0.2%), sodium azide, NP-40 (>1%) and Tween-20 (>1%). Serum and plasma samples should be diluted at least 200× with dH2O. Samples containing higher than 100uM pyruvate (final concentration after any dilutions) require an internal standard.
Procedures1. Standard Curve. Prepare 1000ul 40uM L-lactate Premix by mixing 2ul 20mM Standard and 998ul distilled water. For cell culture samples, prepare 1000ul 40uM L-lactate Premix by mixing 2ul 20mM Standard and 998ul culture medium without serum. Dilute standard as follows.
No Premix+H2O or Medium L-lactate (uM)
1 100ul+0ul 40
2 60ul+40ul 24
3 30ul+70ul 12
4 0ul+100ul 0
Transfer 50ul standards into wells of a black 96-well plate. Samples. Add 50ul of each sample to separate wells of a black 96-well plate.
Samples requiring an internal standard, will need two separate reactions:
1) Sample plus Standard and 2) Sample alone. In addition, each plate will need a Water Blank (0uM L-lactate) reaction. For the internal standard first prepare 400ul 250uM L-lactate standard by mixing 5ul 20mM Standard and 395ul dH2O. For the Sample plus Standard well, add 5ul 250uM L-lactate and 45ul sample. For the Sample wells, add 5ul dH2O and 45ul sample. For the Water Blank add 50ul dH2O.
2. Reagent Preparation. Spin the Enzyme tubes briefly before pipetting. For each Sample and Standard well, prepare Working Reagent by mixing 40ul Assay Buffer, 1ul Enzyme A, 1ul Enzyme B, 10ul NAD and 5ul Probe. Fresh reconstitution is recommended.
3. Reaction. Add 50ul Working Reagent per reaction well quickly. Tap plate to mix. Incubate for 60 min at RT protected from light.
4. Read fluorescence lex/em=530/585nm.
CalculationPlot the L-lactate Standard Curve and determine its slope. The L-lactate concentration of the sample is computed as follows:
[L-Lactate] =
FSAMPLE–FBLANK
Slope (uM-1)
× n (uM)
where FSAMPLE and FBLANK are the fluorescence intensity values of the Sample and 0uM L-lactate (Std 4) respectively. Slope is the slope of the standard curve and n is the dilution factor (e.g. n=200 for serum samples). If an internal standard was needed, the sample L-lactate concentration is computed as follows:
[L-Lactate] =
FSAMPLE–FBLANK
× 27.8 (uM)
FSTANDARD–FSAMPLE
where FSAMPLE and FBLANK are the fluorescence intensity values of the Sample and Water Blank respectively and FSTANDARD is the fluorescence intensity value of the Sample plus Standard.
Note: if the sample DF value is higher than the DF for 40uM L-lactate standard or greater than the DF for the internal standard, dilute the sample in water and repeat the assay. Multiply the results by the dilution factor.
Materials Required, But Not ProvidedPipetting (multi-channel) devices. Black, flat bottom 96-well plates and fluorescent plate reader capable of reading at lex/em=530/585 nm.
Important NoteThis product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.


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