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STAT3 anticorps (C-Term)

KO Validated STAT3 Reactivité: Humain WB, IF, IHC (p), ICC Hôte: Lapin Polyclonal unconjugated
N° du produit ABIN2855865
  • Antigène Voir toutes STAT3 Anticorps
    STAT3 (Signal Transducer and Activator of Transcription 3 (Acute-Phase Response Factor) (STAT3))
    Épitope
    • 97
    • 59
    • 26
    • 8
    • 7
    • 7
    • 7
    • 6
    • 5
    • 4
    • 4
    • 3
    • 3
    • 3
    • 3
    • 3
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    C-Term
    Reactivité
    • 290
    • 150
    • 150
    • 38
    • 27
    • 25
    • 24
    • 19
    • 17
    • 6
    • 4
    • 3
    • 3
    • 3
    • 2
    • 2
    • 2
    • 1
    • 1
    Humain
    Hôte
    • 227
    • 72
    • 4
    • 2
    • 1
    • 1
    Lapin
    Clonalité
    • 195
    • 110
    Polyclonal
    Conjugué
    • 169
    • 23
    • 17
    • 11
    • 11
    • 10
    • 9
    • 7
    • 7
    • 7
    • 7
    • 6
    • 3
    • 3
    • 3
    • 3
    • 3
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    Cet anticorp STAT3 est non-conjugé
    Application
    • 223
    • 110
    • 77
    • 59
    • 51
    • 44
    • 41
    • 41
    • 27
    • 23
    • 15
    • 10
    • 8
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    • 3
    • 3
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
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    Western Blotting (WB), Immunofluorescence (IF), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunocytochemistry (ICC)
     Réactivité croisée
    Humain, Souris, Plantes, Rat
    Attributs du produit
    Rabbit Polyclonal antibody to STAT3 (signal transducer and activator of transcription 3 (acute-phase response factor))
    STAT3 antibody [C3], C-term
    Purification
    Purified by antigen-affinity chromatography.
    Classe de qualité
    KO Validated
    Immunogène
    Carrier-protein conjugated synthetic peptide encompassing a sequence within the C-terminus region of human STAT3. The exact sequence is proprietary.
    Isotype
    IgG
  • Indications d'application
    WB: 1:500-1:3000. ICC/IF: 1:100-1:1000. Optimal dilutions/concentrations should be determined by the researcher. Not tested in other applications.
    Commentaires

    Positive Control: Human ESC , OC3

    Validation: Comparison, KO/KD, Orthogonal

    Restrictions
    For Research Use only
  • Validation #104383 (Cleavage Under Targets and Release Using Nuclease)
    'Independent Validation' signe
    by
    Gianluca Zambanini, Anna Nordin and Claudio Cantù; Cantù Lab, Gene Regulation during Development and Disease, Linköping University
    No.
    #104383
    Date
    07.12.2022
    Antigène
    STAT3
    Numéro du lot
    43873
    Application validée
    Cleavage Under Targets and Release Using Nuclease
    Contrôle positif

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

    Contrôle négative

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

    Conclusion

    Passed. ABIN2855865 allows for CUT&RUN targeted profiling of STAT3 in mouse forelimb tissues.

    'Independent Validation' signe
    Validation Images
    Protocole
    Anticorps primaire
    ABIN2855865
    Anticorps secondaire
    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-STAT3 antibody ABIN2855865, 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.
    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

  • Format
    Liquid
    Concentration
    0.48 mg/mL
    Buffer
    1XPBS ( pH 7), 1 % BSA, 20 % Glycerol, 0.025 % ProClin 300
    Agent conservateur
    ProClin
    Précaution d'utilisation
    This product contains ProClin: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Stock
    4 °C,-20 °C
    Stockage commentaire
    Store as concentrated solution. Centrifuge briefly prior to opening vial. For short-term storage (1-2 weeks), store at 4°C. For long-term storage, aliquot and store at -20°C or below. Avoid multiple freeze-thaw cycles.
  • Chen, Liu, Jang, Hsu, Yen, Liu, Chuang, Wang, Fu, Kuo, Chen: "ERK Activation Modulates Cancer Stemness and Motility of a Novel Mouse Oral Squamous Cell Carcinoma Cell Line." dans: Cancers, Vol. 12, Issue 1, (2019) (PubMed).

  • Antigène
    STAT3 (Signal Transducer and Activator of Transcription 3 (Acute-Phase Response Factor) (STAT3))
    Autre désignation
    signal transducer and activator of transcription 3 (STAT3 Produits)
    Synonymes
    anticorps 1110034C02Rik, anticorps AW109958, anticorps Aprf, anticorps APRF, anticorps HIES, anticorps Xstat3, anticorps aprf, anticorps hies, anticorps stat3, anticorps wu:fc15d02, anticorps wu:fl59g06, anticorps z-Stat3, anticorps signal transducer and activator of transcription 3, anticorps signal transduction and activation of transcription 3, anticorps signal transducer and activator of transcription 3, gene 1 L homeolog, anticorps signal transducer and activator of transcription 3 (acute-phase response factor), anticorps STAT3, anticorps stat3, anticorps Stat3, anticorps stat3.1.L
    Sujet
    The protein encoded by this gene is a member of the STAT protein family. In response to cytokines and growth factors, STAT family members are phosphorylated by the receptor associated kinases, and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators. This protein is activated through phosphorylation in response to various cytokines and growth factors including IFNs, EGF, IL5, IL6, HGF, LIF and BMP2. This protein mediates the expression of a variety of genes in response to cell stimuli, and thus plays a key role in many cellular processes such as cell growth and apoptosis. The small GTPase Rac1 has been shown to bind and regulate the activity of this protein. PIAS3 protein is a specific inhibitor of this protein. Three alternatively spliced transcript variants encoding distinct isoforms have been described.

    Cellular Localization: Cytoplasm , Nucleus
    Poids moléculaire
    88 kDa
    ID gène
    6774
    UniProt
    P40763
    Pathways
    Signalistation JAK/STAT, Signalisation RTK, Interferon-gamma Pathway, Neurotrophin Signaling Pathway, Dopaminergic Neurogenesis, Response to Growth Hormone Stimulus, Carbohydrate Homeostasis, Stem Cell Maintenance, Hepatitis C, Protein targeting to Nucleus, Feeding Behaviour, CXCR4-mediated Signaling Events, Signaling of Hepatocyte Growth Factor Receptor
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