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STAT5A 抗体

This anti-STAT5A antibody is a 兔 单克隆 antibody detecting STAT5A in WB, IHC (p) 和 IF (cc). Suitable for 人, 小鼠 和 大鼠. Independently validated for use in Cleavage Under Targets and Release Using Nuclease.
产品编号 ABIN5557532
发货至: 中国

Quick Overview for STAT5A 抗体 (ABIN5557532)

抗原

See all STAT5A 抗体
STAT5A (Signal Transducer and Activator of Transcription 5A (STAT5A))

适用

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人, 小鼠, 大鼠

宿主

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克隆类型

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单克隆

标记

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This STAT5A antibody is un-conjugated

应用范围

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Western Blotting (WB), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunofluorescence (Cultured Cells) (IF (cc))

克隆位点

6E5
  • 交叉反应

    人, 小鼠, 大鼠

    纯化方法

    Purified by Protein A.

    免疫原

    Recombinant human STAT5a protein, around C-terminal 100aa.

    亚型

    IgG
  • 应用备注

    WB 1:300-5000
    IHC-P 1:200-400
    IF(ICC) 1:50-200

    限制

    仅限研究用
  • 生效 #104384 (Cleavage Under Targets and Release Using Nuclease)
    '独立验证'标志
    by
    Gianluca Zambanini, Anna Nordin and Claudio Cantù; Cantù Lab, Gene Regulation during Development and Disease, Linköping University
    No.
    #104384
    日期
    2022.12.07
    抗原
    STAT5A
    Lot Number
    BA7271967
    Method validated
    Cleavage Under Targets and Release Using Nuclease
    Positive Control

    Polyclonal rabbit anti-H3K4me (antibodies-online, ABIN3023251)

    Negative Control

    Polyclonal guinea pig anti-rabbit IgG (antibodies-online, ABIN101961)

    Notes

    Passed. ABIN5557532 allows for CUT&RUN targeting of STAT5A in mouse forelimb tissue.

    '独立验证'标志
    Validation Images
    Full Methods
    Primary Antibody
    ABIN5557532
    Secondary Antibody
    Full Protocol
    • Cell harvest and nuclear extraction
      • Dissect 3 Fore limbs (11.5 DAC) from mouse strain RjOrl:SWISS for each sample.
      • Dissociate the tissue into single cells in TrypLE (Thermo Fisher Scientific) for 15 min at 37 °C.
      • Centrifuge cell solution 5 min at 800 x g at RT.
      • Remove the liquid carefully.
      • Gently resuspend cells in 1 mL of Nuclear Extraction Buffer (20 mM HEPES-KOH pH 8.2, 20% Glycerol, 0.05% IGEPAL, 0.5 mM Spermidine, 10 mM KCl, Roche Complete Protease Inhibitor EDTA-free).
      • Move the solution to a 2 mL centrifuge tube.
      • Pellet the nuclei 800 x g for 5 min.
      • Repeat the NE wash twice for a total of three washes.
      • Resuspend the nuclei in 20 µL NE Buffer per sample.
    • Concanavalin A beads preparation
      • Prepare one 2 mL microcentrifuge tube.
      • Gently resuspend the magnetic Concanavalin A Beads (antibodies-online, ABIN6952467).
      • Pipette 20 µL Con A Beads slurry for each sample into the 2 mL microcentrifuge tube.
      • Place the tube on a magnet stand until the fluid is clear. Remove the liquid carefully.
      • Remove the microcentrifuge tube from the magnetic stand.
      • Pipette 1 mL Binding Buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) into the tube and resuspend ConA beads by gentle pipetting.
      • Spin down the liquid from the lid with a quick pulse in a table-top centrifuge.
      • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
      • Remove the microcentrifuge tube from the magnetic stand.
      • Repeat the wash twice for a total of three washes.
      • Gently resuspend the ConA Beads in a volume of Binding Buffer corresponding to the original volume of bead slurry, i.e. 20 µL per sample.
    • Nuclei immobilization – binding to Concanavalin A beads
      • Carefully vortex the nuclei suspension and add 20 µL of the Con A beads in Binding Buffer to the cell suspension for each sample.
      • Close tube tightly incubates 10 min at 4 °C.
      • Put the 2 mL tube on the magnet stand and when the liquid is clear remove the supernatant.
      • Resuspend the beads in 1 mL of EDTA Wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free, 2mM EDTA).
      • Incubate 5 min at RT.
      • Place the tube on the magnet stand and when the liquid is clear remove the supernatant.
      • Resuspend the beads in 200 µl of Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free) per sample.
    • Primary antibody binding
      • Divide nuclei suspension into separate 200 µL PCR tubes, one for each antibody (150,000 cells per sample).
      • Add 2 µL antibody (anti-STAT5A antibody ABIN5557532, anti-H3K4me positive control antibody ABIN3023251, and guinea pig anti-rabbit IgG negative control antibody ABIN101961) to the respective tube, corresponding to a 1:100 dilution.
      • Incubate at 4 °C ON.
      • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
      • Remove the microcentrifuge tubes from the magnetic stand.
      • Wash with 200 µL of Wash Buffer using a multichannel pipette to accelerate the process.
      • Repeat the wash five times for a total of six washes.
    • pAG-MNase Binding
      • Prepare a 1.5 mL microcentrifuge tube containing 100 µL of pAG mix per sample (100 µL of wash buffer + 58.5 µg pAG-MNase per sample).
      • Place the PCR tubes with the sample on a magnet stand until the fluid is clear. Remove the liquid carefully.
      • Remove tubes from the magnetic stand.
      • Resuspend the beads in 100 µL of pAG-MNase premix.
      • Incubate 30 min at 4 °C.
      • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
      • Remove the microcentrifuge tubes from the magnetic stand.
      • Wash with 200 µL of Wash Buffer using a multichannel pipette to accelerate the process.
      • Repeat the wash five times for a total of six washes.
      • Resuspend in 100 µL of Wash Buffer.
    • MNase digestion and release of pAG-MNase-antibody-chromatin complexes
      • Place PCR tubes on ice and allow to chill.
      • Prepare a 1.5 mL microcentrifuge tube with 102 µl of 2 mM CaCl2 mix per sample (100 µl Wash Buffer + 2 µL 100 mM CaCl2) and let it chill on ice.
      • Always in ice, place the samples on the magnetic rack and when the liquid is clear remove the supernatant.
      • Resuspend the samples in 100 µl of the 2 mM CaCl2 mix and incubate in ice for exactly 30 min.
      • Place the sample on the magnet stand and when the liquid is clear remove the supernatant.
      • Resuspend the sample in 50 µl of 1x Urea STOP Buffer (8.5 M Urea, 100 mM NaCl, 2 mM EGTA, 2 mM EDTA, 0.5% IGEPAL).
      • Incubate the samples 1h at 4°C.
      • Transfer the supernatant containing the pAG-MNase-bound digested chromatin fragments to fresh 200 µl PCR tubes.
    • DNA Clean up
      • Take the Mag-Bind® TotalPure NGS beads (Omega Bio-Tek, M1378-01) from the storage and wait until they are at RT.
      • Add 2x volume of beads to each sample (e.g. 100 µL of beads for 50 µL of sample).
      • Incubate the beads and the sample for 15 min at RT.
      • During incubation prepare fresh EtOH 80%.
      • Place the PCR tubes on a magnet stand and when the liquid is clear remove the supernatant.
      • Add 200 µl of fresh 80% EtOH to the sample without disturbing the beads (Important!!! Do NOT resuspend the beads or remove the tubes from the magnet stand or the sample will be lost).
      • Incubate 30 sec at RT.
      • Remove the EtOH from the sample.
      • Repeat the wash with 80% EtOH.
      • Resuspend the beads in 25 µL of 10 mM Tris-HCl pH 8.2.
      • Incubate the sample for 2 min at RT.
      • Repeat the 2x beads clean up as described before (this time with 50 µL of beads for each sample).
      • Resuspend the beads + DNA in 20 µL of 10 mM Tris-HCl pH 8.2.
    • Library preparation and sequencing
      • Prepare Libraries using KAPA HyperPrep Kit using KAPA Dual-Indexed adapters according to protocol.
      • Sequence samples on an Illumina NextSeq 500 sequencer, using a NextSeq 500/550 High Output Kit v2.5 (75 Cycles), 36 bp PE.
    • Peak calling
      • Trim reads using using bbTools bbduk (BBMap - Bushnell B. - sourceforge.net/projects/bbmap/) to remove adapters, artifacts and repeat sequences.
      • Map aligned reads to the hg38 human genome using bowtie with options -m 1 -v 0 -I 0 -X 500.
      • Use SAMtools to convert SAM files to BAM files and remove duplicates.
      • Use BEDtools genomecov to produce Bedgraph files.
      • Call peaks using SEACR with a 0.001 threshold and the option norm stringent.
    Experimental Notes

    The protocol is published in Zambanini, G. et al. A New CUT&RUN Low Volume-Urea (LoV-U) protocol uncovers Wnt/β-catenin tissue-specific genomic targets. Development (2022). PMID 36355069

  • 状态

    Liquid

    浓度

    1 μg/μL

    缓冲液

    Aqueous buffered solution containing 1xTBS ( pH 7.4), 1 % BSA, 40 %Glycerol and 0.05 % Sodium Azide.

    储存液

    ProClin

    注意事项

    This product contains ProClin: a POISONOUS AND HAZARDOUS SUBSTANCE, which should be handled by trained staff only.

    储存条件

    4 °C,-20 °C

    储存方法

    Shipped at 4°C. Store at -20°C for one year. Avoid repeated freeze/thaw cycles.

    有效期

    12 months
  • 抗原

    STAT5A (Signal Transducer and Activator of Transcription 5A (STAT5A))

    别名

    STAT5a

    背景

    Synonyms: MGF, STAT5, Signal transducer and activator of transcription 5A, STAT5A

    Background: Carries out a dual function: signal transduction and activation of transcription. Mediates cellular responses to the cytokine KITLG/SCF and other growth factors. Mediates cellular responses to ERBB4. May mediate cellular responses to activated FGFR1, FGFR2, FGFR3 and FGFR4. Binds to the GAS element and activates PRL-induced transcription. Regulates the expression of milk proteins during lactation.

    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
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