Thiobarbituric Acid Reactive Substances (TBARS) Colorimetric BioAssay(TM) Kit

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.\n\nKey Features:\nSensitive and accurate. Linear detection range: Colorimetric Assay: 1-30uM, Fluorometric assay 0.1-1.5uM MDA.\n\nApplications:\nDirect Assays: serum, plasma, urine, saliva and other biological samples.\nDrug Discovery/Pharmacology: effects of drugs on TBARS.\n\nKit Contents:\nTBA Reagent: 25ml Standard: 50ul 15mM MDA\n10% Trichloroacetic acid (TCA): 25ml\nStorage conditions. The kit is shipped at room temperature. Store all components at -20 degrees C. Shelf life of six months after receipt.\nPrecautions: 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.\n\nSample Preparation:\nSamples can be kept frozen at -80 degrees 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:\n1. 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.\n2. Add 200ul ice cold 10% TCA to the 100ul of each sample. Incubate for 5 minutes on ice.\n3. 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.\n\nColorimetric Assay Procedure:\nSet up water bath or heat block and adjust the temperature to 100 degrees 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 degrees C for future use.\n1. 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.\nNo 30uM MDA+H2O Vol (ul) MDA (uM)\n1 300ul+0ul 300 30.0\n2 180ul+120ul 300 18.0\n3 90ul+210ul 300 9.0\n4 0ul+300ul 300 0.0\nSamples. Transfer 200ul of each sample into separate tubes.\n2. Color reaction. To each of the standards and samples, add 200ul TBA Reagent. Vortex tubes to mix and incubate at 100 degrees C for 60 min. Cool down tubes to room temperature. Vortex and briefly centrifuge tubes.\n3. Load 100ul in duplicate from each tube to wells of a clear flatbottom 96-well plate. Read OD at 535nm (525 to 545nm).\n\nFluorimetric Assay: Procedure:\nThe fluorescence assay is 20 times more sensitive than the Colorimetric Assay:.\n1. 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.\n2. For color reaction, add 200ul TBA Reagent. Vortex tubes to mix and incubate at 100 degrees C for 60 min. Cool down tubes to room temperature. Vortex and briefly centrifuge tubes.\n3. 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.\n\nCalculation:\nSubtract 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,\n[TBARS] =\nRSample

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SPECIFICATIONS

Catalog Number

T3774-92

Size

100Tests

References

1. Yagi, K. (1976). A simple fluorometric assay for lipoperoxide in blood plasma. Biochem. Res. 15: 212-216.\n2. Satoh, K. (1978). Serum lipid peroxide in cerebrovascular disorder determined by a new colorimetric method. Clin. Chim. Acts 90: 37-43.\n3. Okawa H. et al (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95: 351-358.