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Protokoll
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1. Standard Curve Preparations: Dilute the 100mM 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 nmol/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 (BioVision, Cat.# 1997-25) 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. 7. Calculation: Correct background by subtracting the value derived from the 0 D-lactate control from all standard and sample readings (
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Applikationshinweise
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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 (BioVision, Cat.# 1997-25) 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 ** ** 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. 7. 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 nmol/well vs. OD 450nm . Apply the sample readings to the standard curve. Calculate the D-Lactate concentrations of the test samples: C = La/Sv (nmol/µl, µmol/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
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