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Insulin ELISA 试剂盒

INS 适用: 人, Pig, Cow Colorimetric Sandwich ELISA Plasma, Serum, Tissue Culture Medium
产品编号 ABIN2866580
发货至: 中国
  • 抗原 See all Insulin (INS) ELISA试剂盒
    Insulin (INS)
    适用
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    人, Pig, Cow
    检测方法
    Colorimetric
    实验类型
    Sandwich ELISA
    应用范围
    ELISA
    原理
    The DetectX® Insulin EIA kit is designed to quantitatively measure insulin present in a variety samples and tissue culture media.
    品牌
    DetectX®
    样品类型
    Serum, Plasma, Tissue Culture Medium
    Analytical Method
    Quantitative
    组件
    Clear Coated 96 Well Plate One Plate Clear plastic microplate with break-apart strips coated with monoclonal antibody to human Insulin.
    Human Insulin Standard 40 μL Insulin at 64,000 pg/mL in a special stabilizing solution.
    DetectX® Insulin Conjugate 5 mL An antibody to human Insulin labeled with peroxidase.
    Assay Buffer Concentrate 28 mL A 5X concentrate that should be diluted with deionized or distilled water.
    Wash Buffer Concentrate 30 mL A 20X concentrate that should be diluted with deionized or distilled water.
    TMB Substrate 11 mL
    Stop Solution 5 mL A 1N hydrochloric acid solution. Caustic.
    Plate Sealer 2 each
    试剂未包括
    Distilled or deionized water.
    Polypropylene or glass test tubes.
    Repeater pipet and disposable tips capable of dispensing 100 and 50 μL.
    A microplate washer.
    Colorimetric 96 well microplate reader capable of reading optical density at 450 nm.
    Software for converting raw relative optical density readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting.
    Contact your plate reader manufacturer for details.
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  • 应用备注
    This assay has been validated for human serum, plasma, and tissue culture media (TCM) samples only.
    Samples containing visible particulate should be centrifuged prior to using.
    Due to the highly conserved nature of insulin it is expected that this kit will measure human, bovine and porcine insulin.
    This assay has low or no reactivity to rat or mouse insulin.
    The end user should test this kit for application in their samples.
    板类型
    Pre-coated
    实验流程
    An Insulin standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve.
    Standards or diluted samples are pipetted into a clear mi- crotiter plate coated with a monoclonal antibody to capture insulin present in the sample.
    After a 60 minute incubation, the plate is washed.
    A peroxidase conjugated insulin antibody is added and the plate is again incubated for 30 minutes and washed.
    Substrate is then added to the plate, which reacts with the bound insulin conjugated antibody.
    After a third incubation, the reaction is stopped and the intensity of the generated color is detected in a microtiter plate reader capable of measuring 450 nm wavelength.
    The concentration of the Insulin in the sample is calculated, after making suitable correction for dilution, using software available with most plate readers.
    试剂准备

    Allow the kit reagents to come to room temperature for 30 minutes.
    We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine Insulin concentrations.
    Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit.
    Assay Buffer Dilute Assay Buffer Concentrate 1:5 by adding one part of the concentrate to four parts of deion- ized water.
    Once diluted this is stable at 4 °C for 3 months.
    Wash Buffer Dilute Wash Buffer Concentrate 1:20 by adding one part of the concentrate to nineteen parts of deionized water.
    Once diluted this is stable at room temperature for 3 months.
    Standard Preparation Label test tubes as #1 through #5.
    Pipet 390 μL of Assay Buffer into tubes #1.
    Pipet 200 μL of Assay Buffer into tubes #2 to #5.
    Carefully add 10 μL of the 64,000 pg/mL Insulin standard to tube #1 and vortex completely.
    Take 200 μL of the Insulin solution in tube #1 and add it to tube #2 and vortex completely.
    Repeat the serial dilutions for tubes #3 through #5.
    The concentration of Insulin in the tubes #1 through #5 will be 1,600, 800, 400, 200 and 100 pg/mL.
    Use all Standards within 2 hours of preparation.

    实验流程
    1. Use the plate layout sheet on the back page of the insert to aid in proper sample and standard identification. Determine the number of wells to be used and return unused wells to foil pouch with desiccant. Seal the ziploc plate bag and store at 4ºC. Pipet standards or samples down the plate strip columns (A to H) to ensure maximum use of the strip wells.
      2. Pipet 50 μL of samples or standards into wells in the plate. Pipet 50 μL of Assay Buffer into the zero standard wells.
      3. Cover the plate with the plate sealer and shake at room temperature for 60 minutes. NOTE: Non-shaking reduces signal by ~ 30 % .
      4. Aspirate the plate and wash each well 4 times with 300 μL wash buffer. Tap the plate dry on clean absorbent towels.
      5. Add 50 μL of the DetectX® Insulin Conjugate to each well, using a repeater pipet.
      6. Cover the plate with the plate sealer and shake at room temperature for 30 minutes.
      7. Aspirate the plate and wash each well 4 times with 300 μL wash buffer. Tap the plate dry on clean absorbent towels.
      8. Add 100 μL of the TMB Substrate to each well, using a repeater pipet.
      9. Incubate the plate at room temperature for 30 minutes, without shaking.
      10. Add 50 μL of the Stop Solution to each well, using a repeater pipet.
      11. Read the optical density generated from each well at 450 nm.
      12. Use the plate reader's built-in 4PLC software capabilities to calculate Insulin concentration for each sample. NOTE: At the end of the assay throw away the used wells and retain the plate frame for use with any remaining wells.
    结果分析

    Average the duplicate OD readings for each standard and sample.
    Create a standard curve by reducing the data using computer software capable of generating a four-parameter logistic curve (4PLC) fit.
    The sample concentrations should be multiplied by the dilution factor to obtain neat sample values.
    Or use the online tool
    tyPical data Sample Mean OD Insulin Conc. (pg/mL) Standard 1 1.493 1,600 Standard 2 0.600 800 Standard 3 0.264 400 Standard 4 0.138 200 Standard 5 0.110 100 Zero 0.081 0 Sample 1 0.466 655.0 Sample 2 0.227 346.6 Always run your own standard curve for calculation of results.
    Do not use this data. 43.33 pg/mL human Insulin is equal to 1 μIU/mL 1 ng of human Insulin is equivalent to 0.023 milliunits of the WHO 1st International Standard 1975 (66/304)

    限制
    仅限研究用
  • 注意事项
    As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
    The complete insert should be read and understood before attempting to use the product.
    The antibody coated plate needs to be stored desiccated.
    The silica gel pack included in the foil ziploc bag will keep the plate dry.
    The silica gel pack will turn from blue to pink if the ziploc has not been closed properly.
    This kit utilizes a peroxidase-based readout system.
    Buffers, including other manufacturers Wash Buffers, containing sodium azide will inhibit color production from the enzyme.
    Make sure all buffers used for samples are azide free.
    Ensure that any plate washing system is rinsed well with deionized water prior to using the supplied Wash Buffer as prepared on Page 8.
    The Stop Solution is acid.
    The solution should not come in contact with skin or eyes.
    Take appro- priate precautions when handling this reagent.
    储存条件
    4 °C,RT
    储存方法
    Once opened the kit can be stored at 4°C up to the expiration date on the kit label.
  • 抗原 See all Insulin (INS) ELISA试剂盒
    Insulin (INS)
    别名
    Insulin (INS 产品)
    别名
    IDDM2 ELISA Kit, ILPR ELISA Kit, IRDN ELISA Kit, MODY10 ELISA Kit, ins1 ELISA Kit, xins ELISA Kit, ins1-a ELISA Kit, Insulin ELISA Kit, AA986540 ELISA Kit, Ins-2 ELISA Kit, InsII ELISA Kit, Mody ELISA Kit, Mody4 ELISA Kit, proinsulin ELISA Kit, zgc:109842 ELISA Kit, igf2-A ELISA Kit, ins ELISA Kit, ins-a ELISA Kit, ins-b ELISA Kit, insulin ELISA Kit, insulin precursor ELISA Kit, insulin II ELISA Kit, preproinsulin ELISA Kit, insulin L homeolog ELISA Kit, insulin S homeolog ELISA Kit, INS ELISA Kit, INS-IGF2 ELISA Kit, ins ELISA Kit, Ins ELISA Kit, PIN ELISA Kit, Ins2 ELISA Kit, ins.L ELISA Kit, ins.S ELISA Kit
    背景
    The human insulin protein is a 51 amino acid anabolic peptide-hormone that is secreted by the pancreatic ß-cells in the Islets of Langerhans. Insulin consists of two chains (A and B) connected by disulfide bonds (1). One of its primary functions is the stimulation of glucose uptake from the systemic circulation, as well as the suppression of hepatic gluconeogenesis, thereby serving a major role in glucose homeostasis and preventing the metabolic disorder diabetes mellitus. The work of Banting, Best, Collip and MacCleod in the early 1920's resulted in the identification of a substance in extracts of pancreas that had the remarkable ability to reduce blood glucose levels in diabetic animals (2) and by 1923 these pancreas extracts were being used to successfully treat diabetic patients. Insulin exists primarily as a monomer at low concentrations (~10-6 M) and forms dimers at higher concentrations at neutral pH (3). At high concentrations and in the presence of zinc ions insulin aggregates further to form hexameric complexes (4). Preproinsulin, the first translational product from the insulin gene, is a 110 amino acid polypeptide with a 24 amino acid signal peptide. Insulin Hexamer The major function of insulin is to counter the concerted actions of a number of hyperglycemia- generating hormones and to maintain low blood glucose levels. In addition to its role in regulating glucose metabolism, insulin stimulates lipogenesis, diminishes lipolysis, and increases amino acid transport into cells. Because there are numerous hyperglycemic hormones, untreated disorders associated with insulin generally lead to severe hyperglycemia and shortened life span. Insu- lin also exerts activities typically associated with growth factors. Insulin is a member of a fam- ily of structurally and functionally similar molecules that include the insulin-like growth factors and relaxin. The tertiary structure of all four molecules is similar, and all have growth-promoting activities. Insulin modulates transcription and stimulates protein translocation, cell growth, DNA synthesis, and cell replication, effects that it holds in common with the insulin-like growth factors and relaxin (see reviews: 5,6)
    途径
    NF-kappaB Signaling, RTK signaling, Positive Regulation of Peptide Hormone Secretion, Peptide Hormone Metabolism, Hormone Activity, Carbohydrate Homeostasis, ER-Nucleus Signaling, Regulation of Carbohydrate Metabolic Process, Feeding Behaviour, Autophagy, Negative Regulation of intrinsic apoptotic Signaling, Brown Fat Cell Differentiation, Positive Regulation of fat Cell Differentiation
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