- 中文名称
Thiobarbituric Acid Reactive Substances (TBARS) Colorimetric BioAssay Kit
- 英文名字
- Thiobarbituric Acid Reactive Substances (TBARS) Colorimetric BioAssay Kit
- 供应商
- USBiological
- 产品货号
- T3774-92
- 产品报价
- ￥1/100Tests
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- 背景资料
- 产品描述
- Oxidative attack of essential cell components by reactive oxygen species has been associated with several human diseases, such as atherosclerosis, cardiovascular diseases, diabetes, liver disorders, and inflammatory rheumatic diseases. Thiobarbituric Acid Reactive Substances (TBARS) are low-molecular-weight end products (mainly malondialdehyde, MDA) that are formed during the decomposition of lipid peroxidation products. Increased levels of TBARS have been demonstrated in these diseases. Simple, direct and accurate assays for TBARS find wide applications in research and drug discovery. TBARS assay is based on the reaction of TBARS with thiobarbituric acid (TBA) to form a pink colored product. The color intensity at 535nm or fluorescence intensity at (lex/em=560nm/585nm) is directly proportional to TBARS concentration in the sample.Key Features:Sensitive and accurate. Linear detection range: Colorimetric Assay: 1-30uM, Fluorometric assay 0.1-1.5uM MDA.Applications:Direct Assays: serum, plasma, urine, saliva and other biological samples.Drug Discovery/Pharmacology: effects of drugs on TBARS.Kit Contents:T3774-92A: TBA Reagent: 25 mL LiquidContains 0.1-1% thiobarbituric acid (CAS #: 504-17-6) and 10-99% dimethylsulfoxide (CAS #: 67-68-5).T3774-92B: Trichloroacetic Acid: 25 mL LiquidContains 10% trichloroacetic acid (CAS #: 76-03-9).T3774-92C: MDA Standard: 50 μL LiquidContains 0.1-0.5% 1,1,3,3-Tetramethoxypropane (CAS #: 102-52-3) and 50-99.9% dimethyl sulfoxide (CAS #: 67-68-5).Storage conditions. The kit is shipped at room temperature. Store all components at -20 °C. Shelf life of six 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:Samples can be kept frozen at -80°C (stable for one month) if not assayed immediately. Urine and saliva samples can be assayed directly (n=1). The following samples need to be deproteinated prior to assay:1. For serum and plasma, transfer 100ul of each sample into a labeled 1.5-mL tube. For tissue samples, weigh ~20 mg into 200ul ice-cold phosphate buffered saline (PBS). Homogenize tissue by brief sonication (e.g. 20 seconds) on ice. If desired, remove 20ul aliquot for protein analysis. Place 100ul tissue lysate into a labeled 1.5ml microcentrifuge tube. For cells, harvest 5 x 106 cells in 200ul ice-cold PBS and sonicate to disrupt cells. If desired, remove 20ul aliquot for protein analysis. Place 100ul cell lysate into a labeled 1.5mL micro-centrifuge tube.2. Add 200ul ice cold 10% TCA to the 100ul of each sample. Incubate for 5 minutes on ice.3. Centrifuge 5 min at 14,000 rpm in an Eppendorf Centrifuge. Transfer 200ul of each clear supernatant into a new labeled tube. Dilution factor for these pretreated samples is n=3.Colorimetric Assay Procedure:Set up water bath or heat block and adjust the temperature to 100°C. Equilibrate all components to room temperature. Add 450ul dH2O to the 15mM Standard tube and mix (final 1.5mM MDA). Store unused Standard at -20°C for future use.1. Standards. Mix 15ul of the 1.5mM MDA with 735ul dH2O (final 30uM MDA). Dilute standards as shown in the Table. Transfer 200ul standards into labeled 1.5-mL screw cap tubes.No 30uM MDA+H2O Vol (ul) MDA (uM)1 300ul+0ul 300 30.02 180ul+120ul 300 18.03 90ul+210ul 300 9.04 0ul+300ul 300 0.0Samples. Transfer 200ul of each sample into separate tubes.2. Color reaction. To each of the standards and samples, add 200ul TBA Reagent. Vortex tubes to mix and incubate at 100°C for 60 min. Cool down tubes to room temperature. Vortex and briefly centrifuge tubes.3. Load 100ul in duplicate from each tube to wells of a clear flatbottom 96-well plate. Read OD at 535nm (525 to 545nm).Fluorometric Assay: Procedure:The fluorescence assay is 20 times more sensitive than the Colorimetric Assay:.1. Prepare the standards as described in the Colorimetric Assay: Procedure:. Transfer 10ul of each Standard into labeled tubes. Add 190ul dH2O (final concentrations 0, 0.45, 0.90, 1.50uM MDA). Samples. In separate tubes, add 200ul of treated samples.2. For color reaction, add 200ul TBA Reagent. Vortex tubes to mix and incubate at 100°C for 60 min. Cool down tubes to room temperature. Vortex and briefly centrifuge tubes.3. Load 100ul in duplicate from each tube to wells of a black flatbottom 96-well plate. Read fluorescence intensity (lex/em=560nm/585nm) on a plate reader.Calculation:Subtract blank OD or fluorescence intensity value (#4) from all standard and sample values. Plot the DOD535nm or DF against standard concentrations and determine the slope of the standard curve. Calculate the TBARS concentration of Sample,[TBARS] =RSample–RBlankSlope (uM-1)× n (uM MDA equivalents)RSAMPLE and RBLANK are the OD535nm or fluorescence intensity values of the sample and H2O blank (standard #4). n is the sample dilution factor (n=3 for deproteinated samples).Note: if calculated TBARS concentration is higher than 30uM MDA (Colorimetric Assay:) or 1.5uM MDA (fluorometric assay) , dilute sample in dH2O and repeat assay. Multiply the results by the dilution factor.Materials Required, But Not Provided:Pipetting devices, centrifuge tubes, centrifuge, clear flat-bottom uncoated 96-well plates, optical density or fluorescence plate readers, sonicator, water-bath or heat block.
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