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ETV4 Antikörper (AA 1-207)

ETV4 Reaktivität: Human WB, IHC, IF Wirt: Kaninchen Polyclonal unconjugated
Produktnummer ABIN6140332
  • Target Alle ETV4 Antikörper anzeigen
    ETV4 (Ets Variant 4 (ETV4))
    Bindungsspezifität
    • 3
    • 3
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    AA 1-207
    Reaktivität
    • 27
    • 22
    • 14
    • 2
    • 2
    • 2
    • 1
    • 1
    Human
    Wirt
    • 24
    • 2
    • 1
    Kaninchen
    Klonalität
    • 25
    • 2
    Polyklonal
    Konjugat
    • 20
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Dieser ETV4 Antikörper ist unkonjugiert
    Applikation
    • 17
    • 9
    • 9
    • 6
    • 5
    • 3
    • 1
    Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence (IF)
    Sequenz
    MYLHTEGFSG PSPGDGAMGY GYEKPLRPFP DDVCVVPEKF EGDIKQEGVG AFREGPPYQR RGALQLWQFL VALLDDPTNA HFIAWTGRGM EFKLIEPEEV ARLWGIQKNR PAMNYDKLSR SLRYYYEKGI MQKVAGERYV YKFVCEPEAL FSLAFPDNQR PALKAEFDRP VSEEDTVPLS HLDESPAYLP ELAGPAQPFG PKGGYSY
    Kreuzreaktivität
    Human, Maus, Ratte
    Produktmerkmale
    Polyclonal Antibodies
    Aufreinigung
    Affinity purification
    Immunogen
    Recombinant fusion protein containing a sequence corresponding to amino acids 1-207 of human Pea3 / ETV4 (NP_001248368.1).
    Isotyp
    IgG
  • Applikationshinweise
    WB,1:500 - 1:2000,IHC,1:50 - 1:200,IF,1:50 - 1:200
    Beschränkungen
    Nur für Forschungszwecke einsetzbar
  • Validierung #104387 (Cleavage Under Targets and Release Using Nuclease)
    'Independent Validation' Siegel
    by
    Gianluca Zambanini, Anna Nordin and Claudio Cantù; Cantù Lab, Gene Regulation during Development and Disease, Linköping University
    No.
    #104387
    Datum
    28.02.2022
    Antigen
    ETV4
    Chargennummer
    BA04368866
    Validierte Anwendung
    Cleavage Under Targets and Release Using Nuclease
    Positivkontrolle

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

    Negativkontrolle

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

    Bewertung

    Passed. ABIN6131217 allows for ETV4 targeted digestion using CUT&RUN in mouse fore limb (11.5) cells.

    'Independent Validation' Siegel
    Validierungsbilder
    Protokoll
    Primärantikörper
    ABIN6131217
    Sekundärantikörper
    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 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.
      • Add 2 µL antibody (anti-ETV4 antibody ABIN6131217, anti-H3K27me3 antibody positive control ABIN6923144, 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.
      • 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.
    • 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.
    Anmerkungen

    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. bioRxiv (2022). https://doi.org/10.1101/2022.07.06.498999

  • Format
    Liquid
    Buffer
    PBS with 0.02 % sodium azide,50 % glycerol, pH 7.3.
    Konservierungsmittel
    Sodium azide
    Vorsichtsmaßnahmen
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Lagerung
    -20 °C
    Informationen zur Lagerung
    Store at -20°C. Avoid freeze / thaw cycles.
  • Target
    ETV4 (Ets Variant 4 (ETV4))
    Andere Bezeichnung
    ETV4 (ETV4 Produkte)
    Synonyme
    E1A-F antikoerper, E1AF antikoerper, PEA3 antikoerper, PEAS3 antikoerper, Pea3 antikoerper, AW414408 antikoerper, Pea-3 antikoerper, pea3 antikoerper, ETS variant 4 antikoerper, ets variant 4 antikoerper, ETV4 antikoerper, Etv4 antikoerper, etv4 antikoerper
    Hintergrund
    Transcriptional activator. May play a role in keratinocyte differentiation.,ETV4,E1A-F,E1AF,PEA3,PEAS3,ETS variant 4,Pea3/ETV4,Epigenetics & Nuclear Signaling,Transcription Factors,Neuroscience,ETV4
    Molekulargewicht
    23 kDa/49 kDa/53 kDa
    Gen-ID
    2118
    UniProt
    P43268
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