D-Lactate Colorimetric Assay Kit

Details for Product No. ABIN411655, Supplier: Log in to see
Antigen
Reactivity
Chemical
3
2
1
Detection Range
0.01-10 mM
Minimum Detection Limit
0.01 mM
Application
Biochemical Assay (BCA)
Options
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Sample Type Cell and Tissue Culture Supernatant, Plasma, Serum, Tissue Samples, Biological Fluids
Detection Method Colorimetric
Specificity D-Lactate Assay Kit provides a fast, easy way to accurately measure D-lactate in a variety of biological samples. In the D-Lactate Assay Kit, D-lactate is specifically oxidized by D-lactate dehydrogenase and generates proportional color (lambda max = 450 nm). The kit detects D-Lactate in samples such as serum, plasma, cells, culture and fermentation media. The useful concentration range in samples is 0.01 mM - 10 mM D-lactate.
Characteristics D-Lactate Colorimetric Assay Kit: Colorimetric Assay to Measure D-Lactate in a variety of Biological Samples such as Cells, Tissues etc. within 40 min. Rapid, Simple & Sensitive.
Components D-Lactate Assay Buffer
D-Lactate Enzyme Mix
D-Lactate Substrate Mix
D-Lactate Standard (100 mM)
Target Type Chemical
Background D-Lactate production in mammals, mainly due to the glyoxalase pathway, is extremely low, with normal serum concentrations in the nano to micromolar range. Typically, elevated D-lactate levels which can rise to millimolar levels, are due to bacterial infection or short bowel syndrome in humans. Abnormally high concentrations of D-lactate are considered indicative of sepsis, ischemia or trauma. Due to slow metabolism and excretion, high D-lactate can cause acidosis and encephalopathy.
Application Notes The useful concentration range in samples is 0.01 mM - 10 mM D-lactate.
Comment

Further details regarding sample type: Cell and tissue culture supernatants, urine, plasma and serum, as well as many other biological fluids, fermentation media

Assay Time < 1 h
Protocol 1. Standard Curve Preparations: Dilute the 100 mM D-Lactate Standard to 1 mM by adding 10 µL of the Standard to 990 µL of Assay Buffer, mix well. Add 0, 2, 4, 6, 8, 10 µL into a series of wells. Adjust volume to 50 µL/well with Assay Buffer to generate 0, 2, 4, 6, 8, 10 nM/well of the D-Lactate Standard.
2. Sample Preparation: Prepare 1-50 µL test samples in a 96-well plate. Adjust the volume to 50 µL /well with Assay Buffer. We suggest using several doses of your sample to ensure the readings are within the standard curve range. Note: (1) Tissue (20 mg) or cells (2 x 10^6 ) can be homogenized in 100 µL the Assay Buffer. Centrifuge at 10,000g for 10 min to remove insoluble materials. The soluble fraction may be assayed directly. (2) Endogenous enzyme activity may cause loss of D-lactate. Samples containing enzyme activity (such as culture medium or tissue lysate) should be kept at -80 °C for storage, or filtered through a 10Kd mw spin filter to remove all proteins.
3. Reaction Mix Preparation: Mix sufficient reagents for the number of assays performed. For each well, prepare a total 50 µL Reaction Mix containing the following components. Mix well before use: 46 µL D-Lactate Assay Buffer 2 µL D-Lactate Substrate Mix 2 µL D-Lactate Enzyme Mix ** ** Note: NADH or NADPH from cell or tissue extracts generates background for the lactate assay. To subtract the NADH or NADPH background, same amount of sample can be tested in the absence of Enzyme Mix, which detect NAD(P)H, not D-Latate. Then the background readings can be subtracted from the D-lactate reading.
4. Add 50 µL of the Reaction Mix to each well containing the D-Lactate Standard or test samples, mix well.
5. Incubate the reaction for 30 minutes at room temperature.
6. Measure O.D. 450 nm in a microplate reader. The color is stable for at least 4 hours.
Calculation of Results

Calculation: Correct background by subtracting the value derived from the 0 D-lactate control from all standard and sample readings (Background can be significant and must be subtracted from all readings). Plot a standard curve of nM/well vs. OD 450nm. Apply the sample readings to the standard curve. Calculate the D-Lactate concentrations of the test samples: C = La/Sv (nM/µL, µM/mL or mM) Where: La is the D-lactate amount (nmol) of your sample from the standard curve. Sv is the sample volume (µL) added into the well. D-Lactic acid molecular weight: 90.08

Restrictions For Research Use only
Storage -20 °C
Expiry Date 12 months
Supplier Images
Functional Studies (Func) image for D-Lactate Colorimetric Assay Kit (ABIN411655) D-Lactate Colorimetric Assay Kit
Product cited in: Wu, Xiao, Ren, Yin, Tan, Liu, Li, Nyachoti, Xiong, Wu: "Therapeutic effects of glutamic acid in piglets challenged with deoxynivalenol." in: PLoS ONE, Vol. 9, Issue 7, pp. e100591, 2014 (PubMed).

Goldgof, Xiao, Chanturiya, Jou, Gavrilova, Reitman: "The chemical uncoupler 2,4-dinitrophenol (DNP) protects against diet-induced obesity and improves energy homeostasis in mice at thermoneutrality." in: The Journal of biological chemistry, Vol. 289, Issue 28, pp. 19341-50, 2014 (PubMed).

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