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NPPA ELISA 试剂盒

NPPA 适用: 小鼠, 人, 大鼠 Colorimetric Competition ELISA 0.1-1.000 pg/mL Cell Culture Supernatant, Serum
产品编号 ABIN1979143
发货至: 中国
  • 抗原 See all NPPA ELISA试剂盒
    NPPA (Natriuretic Peptide A (NPPA))
    适用
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    • 3
    • 3
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    • 1
    • 1
    小鼠, 人, 大鼠
    检测方法
    Colorimetric
    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
    原理
    Human/Mouse/Rat Atrial Natriuretic Peptide EIA Kit optimized for serum and cell culture medium. Competition-based ELISA on a 96-well strip plate.
    样品类型
    Cell Culture Supernatant, Serum
    Analytical Method
    Quantitative
    特异性
    This kit targets the common sequence of human, mouse and rat, and thus may be used to detect ANP expression in all these species with high specificity and sensitivity.

    Cross Reactivity: This EIA kit shows no cross-reactivity with any of the cytokines tested: Ghrelin, Nesfatin, Angiotensin II, NPY and APC.
    交叉反应 (详细)
    This ELISA kit shows no cross-reactivity with any of the cytokines tested: Ghrelin, Nesfatin, Angiotensin II, NPY and APC.
    灵敏度
    1.02 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)
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  • 应用备注
    Recommended Dilution for serum and plasma samplesHuman: 4X / Mouse: 4X / Rat: 4X
    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 ANP Antibody vial (Item N) and reconstitute with 5 µL of ddH2O 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-ANP 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 ANP peptide (Item F) and reconstitute with 20 µL of ddH2O before use. Add 5 µL of Item F to 5 mL 1X Assay Diluent E. Pipette up and down to mix gently. The final concentration of biotinylated ANP will be 10 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: 1000 pg/mL, 100 pg/mL, 10 pg/mL, 1 pg/mL, 0.1 pg/mL and 0 pg/mL. Pipette 450 mL of biotinylated ANP solution into each tube, except for the 1000 pg/mL (leave this one empty). It is very important to make sure the concentration of biotinylated ANP is 10 pg/mL in all standards. a. Briefly centrifuge the vial of standard ANP peptide (Item C) and reconstitute with 10 µL of ddH2O. In the tube labeled 1000 pg/mL, pipette 8 µL of Item C and 792 µL of 10 pg/mL biotinylated ANP solution (prepared in step 4 above). This is your ANP stock solution (1000 pg/mL ANP, 10 pg/mL biotinylated ANP). Mix thoroughly. This solution serves as the first standard. b. To make the 100 pg/mL standard, pipette 50 µL of ANP stock solution the tube labeled 100 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 ANP and 50 mL of the prior concentration until 0.1 pg/mL is reached. Mix each tube thoroughly before the next transfer. d. The final tube (0 pg/mL ANP, 10 pg/mL biotinylated ANP) 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 1X Assay Diluent E. This solution will be used in steps 7 and
      9.
      7. Positive Control Preparation: Briefly centrifuge the positive control vial and reconstitute with 100 µL of ddH2O before use (Item M). To the tube of Item M, add 101 µL 1x Assay Diluent E. Also add 2 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 ANP is 10 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.
      9. Sample Preparation: Use 1X Assay Diluent E + biotinylated ANP to dilute samples, including serum/plasma, cell culture medium and other sample types. 1000 100 10 1 0.1 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL 50 mL 50 mL 50 mL 50 mL It is very important to make sure the final concentration of the biotinylated ANP is 10 pg/mL in every sample.
      Example: to make a 4-fold dilution of sample, mix together 2.5 µL of 10-fold diluted Item F (prepared in step 6), 185 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 ANP to a final concentration of 10 pg/mL.
      Example: Add 2.5 mL of 10-fold diluted Item F to 247.5 mL of sample.
      10. Briefly centrifuge the HRP-Streptavidin vial (Item G) before use. The HRP-Streptavidin concentrate should be diluted 50- fold with 1X Assay Diluent E.
    样品制备

    Use 1X Assay Diluent E + biotinylated ANP to dilute samples, including serum/plasma, cell culture medium and other sample types. 1000 100 10 1 0.1 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL 50 mL 50 mL 50 mL 50 mL It is very important to make sure the final concentration of the biotinylated ANP is 10 pg/mL in every sample. EXAMPLE: to make a 4-fold dilution of sample, mix together 2.5 µL of 10-fold diluted Item F (prepared in step 6), 185 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 ANP to a final concentration of 10 pg/mL. EXAMPLE: Add 2.5 mL of 10-fold diluted Item F to 247.5 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-ANP 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.
      3. Discard the solution and wash wells 4 times with 1x Wash Buffer (200-300 µL each), Washing may be done with a 0 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. 1
    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 Atrial natriuretic 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
  • Shah, Kulkarni, Ferris, Amiji: "Analgesic Efficacy and Safety of DALDA Peptide Analog Delivery to the Brain Using Oil-in-Water Nanoemulsion Formulation." in: Pharmaceutical research, (2014) (PubMed).

    Potter, Yoder, Flora, Antos, Dickey: "Natriuretic peptides: their structures, receptors, physiologic functions and therapeutic applications." in: Handbook of experimental pharmacology, Issue 191, pp. 341-66, (2008) (PubMed).

    Tervonen, Arjamaa, Kokkonen, Ruskoaho, Vuolteenaho: "A novel cardiac hormone related to A-, B- and C-type natriuretic peptides." in: Endocrinology, Vol. 139, Issue 9, pp. 4021-5, (1998) (PubMed).

    Kiberd, Larson, Robertson, Jamison: "Effect of atrial natriuretic peptide on vasa recta blood flow in the rat." in: The American journal of physiology, Vol. 252, Issue 6 Pt 2, pp. F1112-7, (1987) (PubMed).

  • 抗原 See all NPPA ELISA试剂盒
    NPPA (Natriuretic Peptide A (NPPA))
    别名
    ANP (NPPA 产品)
    别名
    anp ELISA Kit, ANF ELISA Kit, ANP ELISA Kit, Pnd ELISA Kit, RATANF ELISA Kit, ATFB6 ELISA Kit, CDD-ANF ELISA Kit, PND ELISA Kit, NPPA ELISA Kit, Anf ELISA Kit, atrial natriuretic peptide ELISA Kit, natriuretic peptide A ELISA Kit, natriuretic peptide type A ELISA Kit, anp ELISA Kit, nppa ELISA Kit, NPPA ELISA Kit, Nppa ELISA Kit
    背景
    Atrial natriuretic peptide (ANP) is a 28-amino acid peptide hormone secreted by cardiac myocytes of the atrium. ANP plays an important role in the homeostatic regulation of body water, sodium, potassium and fat, by acting to reduce the water, sodium and adipose loads on the circulatory system, thus reducing blood pressure. ANP peptide contains a 17-amino acid ring which is formed by a disulfide bond between two cysteine residues at positions 7 and 23. ANP is closely related to BNP (brain natriuretic peptide) and CNP (C- type natriuretic peptide), which all share the same amino acid ring. The mechanism of ANP-induced vasodilatation is through binding to a specific set of ANP receptors. Receptor-agonist binding causes a reduction in blood volume and therefore a reduction in cardiac output and systemic blood pressure. Lipolysis is increased and renal sodium reabsorption is decreased. The overall effect of ANP on the body is to counter increases in blood pressure and volume caused by the renin- angiotensin system. In addition to its vasodilatation effect, ANP also serves as a adipokine. Studies have shown that ANP Increases the release of free fatty acids from adipose tissue, activates adipocyte plasma membrane NPR-A receptors, and increases intracellular cGMP levels that induce the phosphorylation of a hormone-sensitive lipase and perilipin A.
    基因ID
    230899
    UniProt
    P05125
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