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- 应用范围
- Biochemical Assay (BCA)
- 原理
- Total Bile Acid Assay Kit measures the total bile acid within serum, plasma, and cell or tissue lysate samples.
- 样品类型
- Plasma, Cell Samples, Serum, Tissue Lysate
- 产品特性
- Total Bile Acid Assay Kit is a simple colorimetric assay that measures the amount of total bile acid present in plasma, serum, tissue homogenates, or cell lysates in a 96-well microtiter plate format. Each kit provides sufficient reagents to perform up to 100 assays, including blanks, bile acid standards and unknown samples. Sample bile acid concentrations are determined by comparison with a known bile acid standard.
- 组件
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- Bile Acid Standard : One 200 μL vial of a 250 μM glycochenodeoxycholic acid solution in water.
- Assay Reagent A : One 30 mL amber bottle containing thio-NAD+.
- Assay Reagent B : Three 2 mL vials containing 3α-HSD and NADH.
- NADH Reagent : Three 2 mL vials containing NADH.
- 试剂未包括
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- 96 well plate or 96 well strips
- Distilled or deionized water
- 1X PBS
- 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
- 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
- Multichannel micropipette reservoir
- Microplate Spectrophotometer
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Tumor Necrosis Factor alpha (TNF alpha) ELISA Kit
TNF alpha 适用: 大鼠 Colorimetric Sandwich ELISA 15.6 pg/mL - 1000 pg/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
Interleukin 1, beta (IL1B) ELISA KitIL1B 适用: 大鼠 Colorimetric Sandwich ELISA 15.6 pg/mL - 1000 pg/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
Tumor Necrosis Factor alpha (TNF alpha) ELISA KitTNF alpha 适用: 小鼠 Colorimetric Sandwich ELISA 15.6 pg/mL - 1000 pg/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
High Mobility Group Box 1 (HMGB1) ELISA KitHMGB1 适用: 人 Colorimetric Sandwich ELISA 62.5 pg/mL - 4000 pg/mL Plasma, Serum
Amyloid beta 1-40 (Abeta 1-40) ELISA KitAbeta 1-40 适用: 人 Colorimetric Sandwich ELISA 7.813 pg/mL - 500 pg/mL Plasma, Serum, Tissue Homogenate
Amyloid beta 1-42 (Abeta 1-42) ELISA KitAbeta 1-42 适用: 人 Colorimetric Sandwich ELISA 4.688 pg/mL - 300 pg/mL Plasma, Serum, Tissue Homogenate
Lipopolysaccharides (LPS) ELISA Kit适用: Various Species Colorimetric Competition ELISA 12.35 ng/mL - 1000 ng/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
High Mobility Group Box 1 (HMGB1) ELISA KitHMGB1 适用: 小鼠 Colorimetric Sandwich ELISA 46.88 pg/mL - 3000 pg/mL Plasma, Serum
Tumor Necrosis Factor alpha (TNF alpha) ELISA KitTNF alpha 适用: 人 AA 77-233 Colorimetric Sandwich ELISA 15.6 pg/mL - 1000 pg/mL Cell Culture Supernatant, Plasma (EDTA), Plasma (citrate), Plasma (heparin), Serum
Vascular Cell Adhesion Molecule 1 (VCAM1) ELISA KitVCAM1 适用: 人 Colorimetric Sandwich ELISA 1.563-100 ng/mL Plasma, Serum, Tissue Homogenate
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- 应用备注
- Optimal working dilution should be determined by the investigator.
- 说明
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- Measures total bile acid content as low as 1 μM
- Suitable for plasma, serum, and cell or tissue lysates
- Requires absorbance readings at 405 and 630 nm
- 实验流程
- The assay is based on an enzyme driven reaction: when bile acids are incubated in the presence of 3α-hydroxysteroid dehydrogenase (3α-HSD), NADH, and thio-NAD+, thio-NAD+ is converted to its reduced form Thio-NADH. Thio-NADH is then detected colorimetrically as an absorbance increase at 405 nm .
- 样品制备
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Samples should be assayed immediately or stored at -80 °C prior to performing the assay. Optimal experimental conditions for samples must be determined by the investigator. The following recommendations are only guidelines and may be altered to optimize or complement the user's experimental design. A set of serial dilutions is recommended for samples to achieve optimal assay results and minimize possible interfering compounds. Run proper controls as necessary. Always run a standard curve with samples.
- Tissue Lysates: Sonicate or homogenize tissue sample in cold PBS and centrifuge at 10,000 x g for 10 minutes at 4 °C. Aliquot the supernatant for storage at -80 °C. Perform dilutions in deionized H2O.
- Cell Lysates: Wash cells 3 times with cold PBS prior to lysis. Lyse cells with sonication or homogenation in cold PBS and centrifuge at 10,000 x g for 10 minutes at 4 °C. Aliquot the supernatant for storage at -80 °C. Perform dilutions in deionized H2O.
- Serum: Avoid hemolyzed and lipemic blood samples. Collect blood in a tube with no anticoagulant. Allow the blood to clot at room temperature for 30 minutes. Centrifuge at 2500 x g for 20 minutes. Remove the yellow serum supernatant without disturbing the white buffy layer. Aliquot samples for testing and store at -80 °C. Perform dilutions in deionized H2O as necessary.
- Plasma: Avoid hemolyzed and lipemic blood samples. Collect blood with heparin or citrate and centrifuge at 2000 x g and 4 °C for 10 minutes. Remove the plasma layer and store on ice. Avoid disturbing the white buffy layer. Aliquot samples for testing and store at -80 °C. Perform dilutions in deionized H2O as necessary.
- 实验流程
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Each Bile Acid standard and sample should be assayed in duplicate or triplicate. A freshly prepared standard curve should be used each time the assay is performed. 4 Note: Each standard or sample replicate requires two paired wells, one to be treated with 3α-HSD (Reagent B) and one without the enzyme (NADH).
- Add 20 μL of the diluted bile acid standards or samples to the 96-well microtiter plate.
- Add 150 μL of Assay Reagent A to each well and mix contents thoroughly.
- Incubate at 37 °C for 5 minutes.
- Add 50 μL of NADH Reagent to one half of the paired standard or sample wells and mix the well contents thoroughly.
- Add 50 μL of Assay Reagent B to the other half of the paired wells and mix thoroughly.
- Incubate at room temperature for 30 minutes on an orbital shaker.
- Read the plate at a primary wavelength of 405 nm and a secondary wavelength 630 nm using a microplate spectrophotometer.
- 结果分析
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- Subtract the 630 nm absorbance from the 405 nm absorbance.
- Determine the average absorbance values for each sample, control, and standard.
- Subtract the average zero standard value from itself and all standard values.
- Graph the standard curve (see Figure 2).
- Subtract the sample well values without 3α-HSD (NADH) from the sample well values containing enzyme (Assay Reagent B) to obtain the difference. The absorbance difference is due to the enzyme 3α-HSD activity: ΔA = ARgt B - ANADH
- Compare the change in absorbance ?A of each sample to the standard curve to determine and extrapolate the quantity of bile acid present in the sample. Only use values within the range of the standard curve. 6
- 限制
- 仅限研究用
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- 注意事项
- Avoid multiple freeze/thaw cycles.
- 储存条件
- -80 °C
- 储存方法
- Upon receipt, store Assay Reagent A at -20°C. Store all other components at -80°C. If the kit will be used in multiple experiments, aliquot each component before freezing to avoid multiple freeze-thaw cycles. 3
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: "Prolonged feeding with green tea polyphenols exacerbates cholesterol-induced fatty liver disease in mice." in: Molecular nutrition & food research, Vol. 60, Issue 12, pp. 2542-2553, (2016) (PubMed).
: "MitoNEET Deficiency Alleviates Experimental Alcoholic Steatohepatitis in Mice by Stimulating Endocrine Adiponectin-Fgf15 Axis." in: The Journal of biological chemistry, Vol. 291, Issue 43, pp. 22482-22495, (2016) (PubMed).
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: "Prolonged feeding with green tea polyphenols exacerbates cholesterol-induced fatty liver disease in mice." in: Molecular nutrition & food research, Vol. 60, Issue 12, pp. 2542-2553, (2016) (PubMed).
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- 背景
- Bile is a complex mixture of lipids, protein, carbohydrates, mineral salts, vitamins, and various trace elements, with bile acids making up about 67 % of the total composition. Bile acids are produced from excess cholesterol, secreted from the liver, absorbed into the small intestines, and returned to the liver with portal blood. While bile acid synthesis is critical for the removal of cholesterol from the body, bile acids are also needed for proper uptake of dietary lipids, fat soluble vitamins, and other nutrients into the small intestines. Under physiological conditions, newly synthesized bile acids are conjugated to glycine or taurine to form bile salts, and not much free bile acid is actually found in bile. Determining circulatory levels of bile acids can be used to identify or diagnose certain liver diseases. In addition, elevated serum bile levels have been observed in intrahepatic cholestasis of pregnancy cases.
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