Description
TBARS Assay Kit (BA0060) (BA0060)
The TBARS Assay Kit (SKU: BA0060) offers a simple, direct and accurate method for quantifying thiobarbituric acid reactive substances (TBARS) in biological samples. Oxidative attack of essential cell components by reactive oxygen species has been associated with several human diseases, including atherosclerosis, cardiovascular disease, diabetes and inflammatory rheumatic diseases. TBARS are low-molecular-weight end products, mainly malondialdehyde (MDA), formed during the decomposition of lipid peroxidation products, and increased levels have been demonstrated in these conditions. The assay is based on the reaction of TBARS with thiobarbituric acid to form a pink coloured product. The colour intensity at 535 nm, or fluorescence intensity at excitation/emission of 530/550 nm, is directly proportional to the TBARS concentration in the sample.
| Product Name: | TBARS Assay Kit (BA0060) |
| SKU: | BA0060 |
| Detection Method: | Colorimetric or Fluorometric |
| Detection Range: | Colorimetric 1 to 30 uM; fluorometric 0.1 to 1.5 uM MDA |
| Sample Type: | Serum, plasma, urine, saliva and other biological samples, tissue and cells |
| Species Reactivity: | All |
| Assay Time: | Approximately 60 minutes colour reaction plus preparation |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Room Temperature |
Quantitative determination of thiobarbituric acid reactive substances (mainly malondialdehyde). TBARS react with thiobarbituric acid to form a pink product measured colorimetrically at 535 nm or fluorometrically at excitation/emission 530/550 nm.
- Sensitive and accurate
- Colorimetric linear detection range 1 to 30 uM MDA
- Fluorometric linear detection range 0.1 to 1.5 uM MDA
- Fluorescence assay approximately twenty times more sensitive than the colorimetric assay
- Direct assays of serum, plasma, urine, saliva and other biological samples
- Drug discovery and pharmacology: effects of drugs on TBARS
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Sample preparation. Urine and saliva samples can be assayed directly (n = 1). Serum, plasma, tissue and cell samples must be deproteinated: add 200 uL ice-cold 10% TCA to 100 uL sample, incubate 5 minutes on ice, centrifuge 5 minutes at 14,000 rpm and transfer 200 uL of clear supernatant (dilution factor n = 3). |
| 2 | Colorimetric standards. Briefly centrifuge the Standard, mix 4 uL 6 M MDA with 2396 uL distilled water (final 10 mM MDA), then prepare 30 uM MDA by mixing 3 uL 10 mM MDA with 997 uL distilled water. Prepare the dilution series in the table and transfer 200 uL of each standard into labelled screw-cap tubes. Transfer 200 uL of each sample into separate tubes. |
| 3 | Colour reaction. Add 200 uL TBA Reagent to each standard and sample, vortex and incubate at 100C for 60 minutes. Cool to room temperature, vortex and briefly centrifuge. |
| 4 | Load 100 uL in duplicate from each tube into a clear flat-bottom 96-well plate and read OD at 535 nm (525-545 nm). |
| 5 | Fluorimetric assay. Prepare standards as above, transfer 10 uL of each standard into labelled tubes and add 190 uL distilled water (final 0, 0.45, 0.90, 1.50 uM MDA). Add 200 uL treated sample in separate tubes, add 200 uL TBA Reagent, incubate at 100C for 60 minutes, cool, then load 100 uL in duplicate into a black plate and read fluorescence at excitation/emission 530/550 nm. |
Subtract the blank value (standard #4) from all standard and sample values. Plot the change in OD at 535 nm or change in fluorescence against standard concentrations and determine the slope. Calculate TBARS = (Rsample - Rblank) / slope x n, where n is the sample dilution factor (n = 3 for deproteinated samples), expressed in uM MDA equivalents.
| Component | Quantity | Storage |
| TBA Reagent | 25 mL | -20C |
| 10% Trichloroacetic acid (TCA) | 25 mL | -20C |
| Standard (6 M MDA) | 50 uL | -20C |
