AGT ELISA 试剂盒 (Angiotensinogen (serpin Peptidase Inhibitor, Clade A, Member 8))

Details for Product AGT ELISA Kit No. ABIN1979314, 供应商: Log in to see
抗原
  • ANHU
  • SERPINA8
  • AI265500
  • AngI
  • AngII
  • Aogen
  • Serpina8
  • ANRT
  • Ang
  • PAT
  • wu:fb62f06
  • wu:fj87b02
  • zgc:111892
  • AGT
  • angt
  • ANGT
  • SPT
  • Spat
  • AI267024
  • Agat
  • angiotensinogen
  • angiotensinogen (serpin peptidase inhibitor, clade A, member 8)
  • alanine-glyoxylate aminotransferase
  • O-6-methylguanine-DNA methyltransferase
  • AGT
  • Agt
  • agt
  • Agxt
  • Mgmt
Alternatives
Human AGT ELISA Kit
适用
人, 小鼠, 大鼠
其他选择
Kits with alternative reactivity to:
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10
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5
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4
4
2
2
2
实验类型
Competition ELISA
检测范围
0.1-1.000 pg/mL
最低检测浓度
0.1 pg/mL
应用范围
ELISA
选项
Supplier
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原理 Human/Mouse/Rat Angiotensin II EIA Kit optimized for serum. Competition-based ELISA on a 96-well strip plate.
样品类型 Serum
Analytical Method Quantitative
检测方法 Colorimetric
特异性 This kit can theoretically detect all active angiotensins, including ANGI, ANGII, ANGIII and ANGIV. However, it does not detect inactive angiotensinogen.

Cross Reactivity: This EIA kit shows no cross-reactivity with any of the cytokines tested: Ghrelin, Nesfatin, NPY and APC.
交叉反应 (详细) This ELISA kit shows no cross-reactivity with any of the cytokines tested: Ghrelin, Nesfatin, NPY and APC. However, it does not detect inactive angiotensinogen.
灵敏度 2.62 pg/mL
产品特性
  • Strip plates and additional reagents allow for use in multiple experiments
  • Quantitative protein detection
  • Establishes normal range
  • The best products for confirmation of antibody array data
组件
  • Pre-Coated 96-well Strip Microplate
  • Wash Buffer
  • Standard Peptide
  • Assay Diluent(s)
  • Biotinylated Peptide
  • HRP-Streptavidin
  • TMB One-Step Substrate
  • Stop Solution
  • Assay Diagram
  • Positive Control Sample
  • Capture Antibody
  • User Manual
试剂未包括
  • Distilled or deionized water
  • Precision pipettes to deliver 2 μL to 1 mL volumes
  • Adjustable 1-25 mL pipettes for reagent preparation
  • 100 mL and 1 liter graduated cylinders
  • Tubes to prepare standard and sample dilutions
  • Orbital shaker
  • Aluminum foil
  • Saran Wrap
  • Absorbent paper
  • Microplate reader capable of measuring absorbance at 450nm
  • SigmaPlot software (or other software that can perform four-parameter logistic regression models)
别名 Angiotensin II, Angiotensinogen (AGT ELISA Kit 摘要)
背景 Angiotensin II has been associated with a number of important physiological processes in heart, brain, adrenal gland and kidney. For cardiovascular effect, Angiotensin II is a potent direct vasoconstrictor, constricting arteries and veins and increasing blood pressure. It is also the most important Gq stimulator of the heart during hypertrophy. For neural effects, Angiotensin II increases thirst sensation (dipsogen) through the subfornical organ (SFO) of the brain, decreases the response of the baroreceptor reflex, and increases the desire for salt. It increases secretion of ADH in the posterior pituitary and secretion of ACTH in the anterior pituitary. For adrenal effects, Angiotensin II acts on the adrenal cortex, causing it to release aldosterone. For renal effects, Angiotensin II has a direct effect on the proximal tubules to increase Na + absorption. Angiotensin, a key player in the renin-angiotensin system, is a peptide hormone that causes vasoconstriction, increased blood pressure, and release of aldosterone from the adrenal cortex. It is derived from the precursor molecule angiotensinogen produced in the liver. Angiotensin II is formed from Angiotensin I, which is removed of two terminal residues by the enzyme Angiotensin-converting enzyme (ACE). Angiotensin II acts as an endocrine, autocrine/ paracrine, and intracrine hormone. Angiotensin II is degraded to angiotensin III by angiotensinases that are located in red blood cells and the vascular beds of most tissues. It has a half-life in circulation of around 30 seconds, while in tissue, it may be as long as 15-30 minutes. The effect of obesity on Angiotensin II has recently been reported.
UniProt P11859
途径 JAK/STAT Signaling, ACE Inhibitor Pathway, EGFR Signaling Pathway, Peptide Hormone Metabolism, Regulation of Systemic Arterial Blood Pressure by Hormones, Regulation of Lipid Metabolism by PPARalpha, Protein targeting to Nucleus, Feeding Behaviour, Monocarboxylic Acid Catabolic Process, Dicarboxylic Acid Transport, Positive Regulation of Response to DNA Damage Stimulus, Regulation of long-term Neuronal Synaptic Plasticity
应用备注 Recommended Dilution for serum and plasma samplesHuman: 2X / Mouse: 2X / Rat: 2X
样本量 100 μL
实验时间 5 h
板类型 Pre-coated
实验流程
  1. Prepare all reagents, samples and standards as instructed.
  2. Add 100 μL detection antibody to each well.
  3. Incubate 1.5 h at RT or O/N at 4 °C.
  4. Add 100 μL standard or sample to each well.
  5. Incubate 2.5 h at RT.
  6. Add 100 μL prepared streptavidin solution.
  7. Incubate 45 min at RT.
  8. Add 100 μL TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 μL Stop Solution to each well.
  11. Read plate at 450 nm immediately.
试剂准备
  1. Keep kit reagents on ice during steps. Equilibrate plate to room temperature before opening the sealed pouch.
    2. Briefly centrifuge the Anti-Angiotensin II Antibody vial (Item N) before use. Add 50 µL of 1x Assay Diluent E into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently.
    3. The antibody concentrate should then be diluted 100-fold with 1x Assay Diluent E. This is your anti-Angiotensin II antibody working solution, which will be used in step 2 of the Assay Procedure. NOTE: the following steps may be done during the antibody incubation procedure (step 2 of Assay Procedure).
    4. Briefly centrifuge the vial of biotinylated Angiotensin II peptide (Item F) before use. Add 10 µL of Item F to 5 mL of the 1X Assay Diluent E. Pipette up and down to mix gently. The final concentration of biotinylated Angiotensin II will be 200 pg/mL. This solution will only be used as the diluent in step 5 of Reagent Preparation.
    5. Preparation of Standards: Label 6 microtubes with the following concentrations: 10000 pg/mL, 1000 pg/mL, 100 pg/mL, 10 pg/mL, 1 pg/mL and 0 pg/mL. Pipette 450 mL of biotinylated Angiotensin II solution into each tube, except for the 10000 pg/mL (leave this one empty). It is very important to make sure the concentration of biotinylated Angiotensin II is 200 pg/mL in all standards. a. Briefly centrifuge the vial of standard Angiotensin II peptide (Item C). In the tube labeled 10000 pg/mL, pipette 8 µL of Item C and 792 µL of 200 pg/mL biotinylated Angiotensin II solution (prepared in step 4 above). This is your Angiotensin II stock solution (10000 pg/mL Angiotensin II, 200 pg/mL biotinylated Angiotensin II). Mix thoroughly. This solution serves as the first standard. b. To make the 1000 pg/mL standard, pipette 50 µL of Angiotensin II stock solution the tube labeled 1000 pg/mL. Mix thoroughly. c. Repeat this step with each successive concentration, preparing a dilution series as shown in the illustration below. Each time, use 450 mL of biotinylated Angiotensin II and 50 mL of the prior concentration until 1 pg/mL is reached. Mix each tube thoroughly before the next transfer. d. The final tube (0 pg/mL Angiotensin II, 200 pg/mL biotinylated Angiotensin II) serves as the zero standard (or total binding).
    6. Prepare a 10-fold dilution of Item F. To do this, add 2 mL of Item F to 18 mL of the 1XAssay Diluent E. This solution will be used in steps 7 and
    9.
    7. Positive Control Preparation: Briefly centrifuge the positive control vial (Item M). To the tube of Item M, add 101 µL 1x Assay Diluent E. Also add 4 µL of 10-fold diluted Item F (prepared in step 6) to the tube. This is a 2-fold dilution of the positive control. Mix thoroughly. The positive control is a cell culture medium sample that is meant to be a system control (to verify that the detection & kit components are working). It may be diluted further if desired, but be sure the final concentration of biotinylated Angiotensin II is 200 pg/mL.
    8. If Item B (20X Wash Concentrate) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to yield 400 mL of 1X Wash Buffer. 10000 1000 100 10 1 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL 50 mL 50 mL 50 mL 50 mL
    9. Sample Preparation: Use 1X Assay Diluent E + biotinylated ANGII to dilute samples, including serum/plasma, cell culture medium and other sample types. It is very important to make sure the final concentration of the biotinylated Angiotensin II is 200 pg/mL in every sample.
    Example: to make a 4-fold dilution of sample, mix together 5 µL of 10-fold diluted Item F (prepared in step 6), 182.5 mL of 1X Assay Diluent E, and 62.5 µL of your sample, mix gently. The total volume is 250 µl, enough for duplicate wells on the microplate. Do not use Item F diluent from Step 5 for sample preparation. If you plan to use undiluted samples, you must still add biotinylated Angiotensin II to a final concentration of 200 pg/mL.
    Example: Add 5 mL of 10-fold diluted Item F to 245 mL of sample.
    10. Briefly centrifuge the HRP-Streptavidin vial (Item G) before use. The HRP-Streptavidin concentrate should be diluted 200- fold with 1X Assay Diluent E.
样品制备

Use 1X Assay Diluent E + biotinylated ANGII to dilute samples, including serum/plasma, cell culture medium and other sample types. It is very important to make sure the final concentration of the biotinylated Angiotensin II is 200 pg/mL in every sample. EXAMPLE: to make a 4-fold dilution of sample, mix together 5 µL of 10-fold diluted Item F (prepared in step 6), 182.5 mL of 1X Assay Diluent E, and 62.5 µL of your sample, mix gently. The total volume is 250 µl, enough for duplicate wells on the microplate. Do not use Item F diluent from Step 5 for sample preparation. If you plan to use undiluted samples, you must still add biotinylated Angiotensin II to a final concentration of 200 pg/mL. EXAMPLE: Add 5 mL of 10-fold diluted Item F to 245 mL of sample.

实验流程
  1. Keep kit reagents on ice during reagent preparation steps. It is recommended that all standards and samples be run at least in duplicate.
    2. Add 100 µL anti-Angiotensin II antibody (see Reagent Preparation step 3) to each well. Incubate for 1.5 hours at room temperature with gentle shaking (1-2 cycles/sec). You may also incubate overnight at 4 degrees C. 0
    3. Discard the solution and wash wells 4 times with 1x Wash Buffer (200-300 µL each). Washing may be done with a multichannel pipette or an automated plate washer. Complete removal of liquid at each step is essential to good assay performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
    4. Add 100 µL of each standard (see Reagent Preparation step 5), positive control (see Reagent Preparation step 7) and sample (see Reagent Preparation step 9) into appropriate wells. Be sure to include a blank well (Assay Diluent only). Cover wells and incubate for 2.5 hours at room temperature with gentle shaking (1-2 cycles/sec) or overnight at 4 °C.
    5. Discard the solution and wash 4 times as directed in Step
    3.
    6. Add 100 µL of prepared HRP-Streptavidin solution (see Reagent Preparation step 10) to each well. Incubate for 45 minutes with gentle shaking at room temperature. It is recommended that the incubation time should not be shorter or longer than 45 minutes.
    7. Discard the solution and wash 4 times as directed in Step
    3.
    8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking (1-2 cycles/sec).
    9. Add 50 µL of Stop Solution (Item I) to each well. Read absorbances at 450 nm immediately.
结果分析

Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the blank optical density. Plot the standard curve using SigmaPlot software (or other software which can perform four-parameter logistic regression models), with standard concentration on the x-axis and percentage of absorbance on the y-axis. Draw the best-fit curve through the standard points.

实验精密度 Intra-Assay: CV < 10 %
Inter-Assay: CV < 15 %
限制 仅限研究用
注意事项 Avoid repeated freeze/thaw cycles.
储存条件 -20 °C
储存方法 Standard, Biotinylated Angiotensin II peptide, and Positive Control should be stored at -20°C after arrival. Avoid multiple freeze-thaws. The remaining kit components may be stored at 4°C. Opened Microplate Wells and antibody (Item N) may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack and reseal along entire edge.
有效期 6 months
有引用在: Shigemura, Iwata, Yasumatsu, Ohkuri, Horio, Sanematsu, Yoshida, Margolskee, Ninomiya: "Angiotensin II modulates salty and sweet taste sensitivities." in: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 33, Issue 15, pp. 6267-77, 2013 (PubMed).

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