Malate Colorimetric Assay Kit Kit
- Detection Method
- Biochemical Assay (BCA)
- Sample Type
- Cell Culture Supernatant, Plasma, Serum, Tissue Samples
- The Kit has developed an easy and sensitive assay to measure the L(-) Malate level in a variety of samples. In the assay, malate is specifically oxidized to generate a product which reacts with a substrate probe to generate color (lambda max = 450 nm). The assay can detect 1 approx. 35 nmol of Malate in a 50 μL sample with a detection sensitivity approx. 20 μM.
- Malate Assay Kit: Colorimetric Assay to Measure L(-) Malate in variety of Samples such as Tissue, Food, Beverage etc. within 40 min.
Malate Assay Buffer
Malate Enzyme Mix
Malate Standard (10 μmol)
- Application Notes
- The assay can detect 1 ~ 35 nmol of Malate in a 50 μL sample with a detection sensitivity ~20 μM.
Further details regarding sample type: Cell and tissue culture supernatants, urine, plasma and serum, as well as many other biological fluids, food, fruits, cheese, beer and wine samples
- Assay Time
- < 1 h
1. Standard Curve Preparations: Dilute the Malate Standard to 2.0 nM/µL by adding 20 µL of the Standard to 980 µL of dH2O, mix well. Add 0, 2.5, 5.0, 7.5, 10.0, 12.5 µL into a series of wells on a 96 well plate. Adjust volume to 50 µL/well with Assay Buffer to generate 0, 5, 10, 15, 20, 25 nM/well of the Standard.
2. Sample Preparation: Tissue samples: (10-100 mg) should be rapidly homogenized with two volumes of ice cold PBS or other buffer (pH 6.5-8). Enzymes in samples may interfere with the assay. We suggest deproteinizing your sample using a perchloric acid/KOH protocol or 10 kd molecular weight cut off spin columns. Add 1-50 µL samples into duplicate wells of a 96-well plate and bring volume to 50 µL with Assay Buffer. We suggest testing several doses of your samples to ensure readings are within the standard curve range. Food or Beverage samples: Most beverages can be used directly in the assay, with appropriate dilution (Beer, no dilution, wine approx. 1:10 dilution). If protein or fat is present, samples should be spin filtered through a 10kD MWCO filter such as The Kit ABIN413915. Solids should be processed by homogenizing 20 mg with 500 µL distilled water, with mild heating for 30 minutes, then centrifuge 10k x g, 10 minutes, take supernates, spin filter and dilute appropriately for the assay.
3. Develop: Mix enough reagent for the number of samples and standards to be performed: For each well, prepare a total 50 µL Reaction Mix containing: Malate Assay Buffer 43 µL Malate Enzyme Mix* 2 µL WST Substrate 5 µL Add 50 µL of the Reaction Mix to each well containing the Malate Standard and test samples. * Note: Some components in samples may generate background in the assay such as NAD(P)H and other reducing agents, etc. If such materials are presence in your samples, you may need to do a background control by omitting the Malate Enzyme Mix in the reaction mix replacing with 2 µL of assay buffer. The background readings should be then subtracted from Malate readings.
4. Incubate for 30 minutes at 37 °C, protect from light.
5. Measure OD at 450 nm in a micro-plate reader.
- Calculation of Results
Calculation: Correct background by subtracting the value of the 0 Malate blank from all standard and sample readings (If sample background controls are generated, subtract the background control readings from malate readings). Plot the standard curve. Then apply the corrected sample readings to the standard curve to get Malate amount in the sample wells. The Malate concentrations in the test samples: C = Ay/Sv (nM/µL, or μM/mL, or mM) Where: Ay is the amount of Malate (nmol) in your sample from the standard curve. Sv is the sample volume (µL) added to the sample well. Malic acid molecular weight: 134.09 L(-) Malate standard curve generated using this kit protocol.
- For Research Use only
- -20 °C
- Expiry Date
- 12 months
Gaglio, Metallo, Gameiro, Hiller, Danna, Balestrieri, Alberghina, Stephanopoulos, Chiaradonna: "Oncogenic K-Ras decouples glucose and glutamine metabolism to support cancer cell growth." in: Molecular systems biology, Vol. 7, pp. 523, 2011 (PubMed).
- Gaglio, Metallo, Gameiro, Hiller, Danna, Balestrieri, Alberghina, Stephanopoulos, Chiaradonna: "Oncogenic K-Ras decouples glucose and glutamine metabolism to support cancer cell growth." in: Molecular systems biology, Vol. 7, pp. 523, 2011 (PubMed).
- Target Type
- L(-) Malate is a TCA cycle intermediate. It plays an important role in the Calvin cycle during carbon fixation in plants. In lower organisms, malate is converted to lactate during malolactic fermentation with the formation of CO₂. Malate is frequently used as an additive in the food and pharmaceutical industries, so quantitating malic acid is important in manufacturing beer, wine, cheese and fruits, among others. BioVision has developed an easy and sensitive assay to measure the L(-) Malate level in a variety of samples. In the assay, malate is specifically oxidized to generate a product which reacts with a substrate probe to generate color (λmax = 450 nm). The assay can detect 1 ~ 35 nmol of Malate in a 50 μL sample with a detection sensitivity ~20 μM.