STAT5A ELISA 试剂盒 (Signal Transducer and Activator of Transcription 5A)

Details for Product STAT5A ELISA Kit No. ABIN1981843, 供应商: Log in to see
抗原
  • STAT5A
  • STAT5
  • MGF
  • AA959963
  • Stat5
  • STAT5A/MGF
  • STATA5
  • signal transducer and activator of transcription 5A
  • signal transducer and activator of transcription 5A-like
  • signal transducer and activator of transcription 5B
  • STAT5A
  • Stat5a
  • LOC100348518
  • STAT5B
Alternatives
Human STAT5A ELISA Kit
抗原表位
pTyr694
适用
人, 小鼠, 大鼠
其他选择
Kits with alternative reactivity to:
26
16
15
3
3
2
2
1
1
1
1
实验类型
Cell ELISA
应用范围
ELISA
选项
Supplier
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Supplier Product No.
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原理 Cell-Based Human Stat 5 (Tyr694) Phosphorylation ELISA Kit. Suitable for adherent whole cell lines.
品牌 CellBIND®
样品类型 Cell Culture Cells
Analytical Method Semi-Quantitative
检测方法 Colorimetric
特异性 The antibodies provided in this kit recognizes human Stat 5 phosphorylated at site Tyrosine-694 and total Stat 5 for comparison.
产品特性
  • Site and signal pathway-specific
  • In vitro detection of adherent cell culture
  • No sample lysis needed
  • Compatible with a standard ELISA plate reader
  • Faster results than with ELISA
  • Adaptable for high-throughput screening and drug discovery
组件
  • uncoated 96-well Microplate
  • Wash Buffer A
  • Wash Buffer B
  • Fixing Solution
  • Quenching Buffer
  • Blocking Buffer
  • Anti-phospho antibody
  • Anti-pan antibody
  • HRP-Conjugated Secondary Antibody
  • TMB One-Step Substrate
  • Stop Solution
  • 试剂未包括
    • Distilled or deionized water
    • 100 mL and 1 liter graduated cylinders
    • Tubes to prepare sample dilutions
    • Protease and Phosphatase inhibitors
    • Precision pipettes to deliver 2 μL to 1 mL volumes
    • Adjustable 1-25 mL pipettes for reagent preparation
    • Benchtop rocker or shaker
    • Microplate reader capable of measuring absorbance at 450 nm
    别名 STAT5 (STAT5A ELISA Kit 摘要)
    背景 STAT5
    基因ID 6776
    UniProt P42229
    途径 JAK/STAT Signaling, RTK signaling, Response to Growth Hormone Stimulus, C21-Steroid Hormone Metabolic Process, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, CXCR4-mediated Signaling Events, Activated T Cell Proliferation
    样本量 100 μL
    板类型 Uncoated
    实验流程
    1. Seed 10,000-30,000 cells into each well and incubate overnight.
    2. Apply various treatment, inhibitors or activators according to manufacture's instructions.
    3. Add 100 μL of Fixing Solution into each well and incubate for 20 min at RT with shaking.
    4. Add 200 μL of prepared 1X Quenching Buffer and incubate 20 min at RT.
    5. Add 200 μL of Blocking Solution and incubate for 1 h at 37 °C.
    6. Add 50 μL of 1X anti-phospho-protein specific antibody or anti-pan-protein specific antibody to each well and incubate for 2 h at RT.
    7. Add 50 μL of prepared 1X HRP-Anti-Rabbit or Mouse IgG and incubate for 1 h at RT.
    8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
    9. Incubate 30 min at RT.
    10. Add 50 μL of Stop Solution to each well.
    11. Read at 450 nm immediately.
    试剂准备

    NOTE: Thaw all reagents to room temperature immediately before use. If wash buffers contain visible crystals, warm to room temperature and mix gently until dissolved.
    NOTE: Briefly centrifuge (~1,000g) ITEMS G, H, and I before opening to ensure maximum recovery.
    For more information look at the picture.

    实验流程

    NOTE: ALL incubations and wash steps must be performed under gentle rocking or rotation (~1-2 cycles/sec).
    1. Design your experiment. For example, see Figure 2 below.
    OPTIONAL: If seeding HUVECs, HMEC-1 or other loosely attached cells, coat the Uncoated 96-Well Microplate (ITEM A) by adding 100 µL poly-L-Lysine (Recommended Sigma Aldrich) into each well and then follow manufacturer's instructions. A pre-coated CellBIND® microplate or other poly-lysine treated tissue culture plate may be used in place of Item A.
    2. Seed 100 µL of 20,000 cells into each well of the Uncoated 96-Well Microplate (ITEM A) provided and incubate overnight at 37 °C with 5 % CO2.
    NOTE: The optimal cell number used will vary on the cell line and the relative amount of protein phosphorylation. More or less cells may be used but this must be determined empirically.
    NOTE: The cells can be starved ~4-24 hours (depending on cell line) prior to treatment with inhibitors or activators.
    3. Apply various treatments, inhibitors (such as siRNA or chemicals) or activators according to manufacturer's instructions and incubate for the desired time points.
    NOTE: It is recommended to dissolve inhibitors or activators into serum-free cell culture medium before treating the cells (unless otherwise stated in the manufacturer's instructions.)
    4. Discard the cell culture medium by flipping the microplate upside down and gently tapping the bottom of the microplate over a sink.
    5. Wash by pipetting 200 µL of the prepared 1X Wash Buffer A (ITEM B) into each well. Discard the wash buffer (same as step 4) and wash 2 more times for a total of 3 washes using fresh wash buffer each time. After the final wash, gently blot the microplate onto a paper towel to remove any excess/remaining buffer.
    NOTE: To avoid cell loss, do not pipette directly onto the cells. Instead, gently dispense the liquid down the wall of cell culture wells. Also avoid the use of vacuum suction or too forcefully tapping the microplate when discarding any solution.
    6. Add 100 µL of Fixing Solution (ITEM D) into each well and incubate for 20 minutes at room temperature.
    NOTE: The fixing solution is used to permeabilize the cells.
    7. Repeat wash step 5.
    8. Add 200 µL of the prepared 1X Quenching Buffer (ITEM E) into each well and incubate 20 minutes at room temperature.
    NOTE: The quenching buffer is used to minimize the background response.
    9. Wash 4 times with 1X Wash Buffer A.
    10. Add 200 µL of the prepared 1X Blocking Buffer (ITEM F) into each well and incubate for 1 hour at 37 °C.
    11. Wash 3 times with the prepared 1X Wash Buffer B (ITEM C).
    NOTE: If needed, the microplate may be stored at -80 °C for several days after this wash.
    12. Add 50 µL of the prepared 1X primary antibody (ITEM G or H) into each corresponding well and incubate for 2 hours at room temperature.
    13. Wash 4 times with 1X Wash Buffer B.
    14. Add 50 µL of the prepared 1X HRP Conjugated secondary antibody (ITEM I) into each well and incubate for 1 hour at room temperature.
    NOTE: The Item I dilution factor will depend on the primary antibody. See Section IV. Reagent Preparation for details.
    15. Wash 4 times with 1X Wash Buffer B.
    16. Add 100 µL of the TMB Substrate (ITEM J) into each well and incubate for 30 minutes at room temperature in the dark.
    17. Add 50 µL of the Stop Solution (ITEM K) into each well. Read at 450 nm immediately.

    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 The entire kit may be stored at -20°C for up to 6 months from the date of shipment. Avoid repeated freeze-thaw cycles.
    有效期 6 months
    厂商提供的图像
     image for Signal Transducer and Activator of Transcription 5A (STAT5A) ELISA Kit (ABIN1981843) Cell-Based protein phosphorylation procedure
     image for Signal Transducer and Activator of Transcription 5A (STAT5A) ELISA Kit (ABIN1981843) This picture shows the reagent preparation.
     image for Signal Transducer and Activator of Transcription 5A (STAT5A) ELISA Kit (ABIN1981843) Example of how to seed cells for cell-based assay
     image for Signal Transducer and Activator of Transcription 5A (STAT5A) ELISA Kit (ABIN1981843) A431 cells were stimulated by different concentrations of EGF for 10 minutes at 37 °C
     image for Signal Transducer and Activator of Transcription 5A (STAT5A) ELISA Kit (ABIN1981843) A431 cells were stimulated by different concentrations of EGF for 30 minutes at 37 °C
    Western Blotting (WB) image for Signal Transducer and Activator of Transcription 5A (STAT5A) ELISA Kit (ABIN1981843) Western blot analysis of extracts from 100 ng/mL hEGF treated A431 cells. Phospho-Sta...
    有引用在: Park, Lee, Frank, Razani, Nguyen, Parlow, Russell, Hulit, Pestell, Lisanti: "Caveolin-1-deficient mice show accelerated mammary gland development during pregnancy, premature lactation, and hyperactivation of the Jak-2/STAT5a signaling cascade." in: Molecular biology of the cell, Vol. 13, Issue 10, pp. 3416-30, 2002

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