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Histone 3 Antikörper (H3K27ac)

H3 Reaktivität: Human, Saccharomyces cerevisiae WB, IF, ChIP, DB, ChIP-seq, ICC, CUT&RUN, CUT&Tag Wirt: Kaninchen Polyclonal unconjugated
Produktnummer ABIN2667903
  • Target Alle Histone 3 (H3) Antikörper anzeigen
    Histone 3 (H3)
    Bindungsspezifität
    • 64
    • 47
    • 46
    • 45
    • 42
    • 37
    • 36
    • 35
    • 35
    • 35
    • 33
    • 32
    • 31
    • 30
    • 29
    • 29
    • 26
    • 25
    • 23
    • 23
    • 23
    • 22
    • 20
    • 18
    • 18
    • 17
    • 17
    • 16
    • 16
    • 16
    • 16
    • 16
    • 15
    • 14
    • 14
    • 13
    • 13
    • 12
    • 12
    • 12
    • 12
    • 12
    • 11
    • 11
    • 10
    • 10
    • 10
    • 9
    • 9
    • 8
    H3K27ac
    Reaktivität
    • 1410
    • 942
    • 862
    • 59
    • 42
    • 41
    • 41
    • 40
    • 39
    • 35
    • 35
    • 30
    • 29
    • 22
    • 19
    • 6
    • 3
    • 3
    • 3
    • 3
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Human, Saccharomyces cerevisiae
    Wirt
    • 1219
    • 248
    • 12
    • 4
    Kaninchen
    Klonalität
    • 1120
    • 362
    • 1
    Polyklonal
    Konjugat
    • 826
    • 81
    • 65
    • 64
    • 63
    • 63
    • 63
    • 62
    • 37
    • 33
    • 24
    • 17
    • 17
    • 17
    • 17
    • 17
    • 14
    • 2
    • 1
    Dieser Histone 3 Antikörper ist unkonjugiert
    Applikation
    • 1061
    • 411
    • 381
    • 316
    • 253
    • 188
    • 186
    • 180
    • 163
    • 160
    • 141
    • 126
    • 119
    • 48
    • 38
    • 34
    • 32
    • 20
    • 15
    • 13
    • 7
    • 7
    • 6
    • 4
    • 3
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Western Blotting (WB), Immunofluorescence (IF), Chromatin Immunoprecipitation (ChIP), Dot Blot (DB), ChIP DNA-Sequencing (ChIP-seq), Immunocytochemistry (ICC), Cleavage Under Targets and Release Using Nuclease (CUT&RUN), Cleavage Under Targets and Tagmentation (CUT&Tag)
    Aufreinigung
    Protein A Chromatography
    Isotyp
    IgG
    Top Product
    Discover our top product H3 Primärantikörper
  • Applikationshinweise
    This rabbit anti-H3K27ac antibody is suitable for use in CUT&RUN, CUT&Tag, ChIP-seq, Chromatin Immunoprecipitation, , Dot Blot, Immunocytochemistry, Immunofluorescence, and Western Blot. Specific conditions for each assay should be optimized by the end user. General dilution recommendations for different applications are as follows:
    CUT&RUN: 1:100
    CUT&Tag: 1:100
    ChIP: 10 µg/ChIP
    ChIP-seq: 5 µg/ChIP
    ICC: 1-5 µg/ml
    IF: 1-5 µg/ml
    WB: 0.1-1 µg/ml
    Beschränkungen
    Nur für Forschungszwecke einsetzbar
  • Validierung #104485 (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.
    #104485
    Datum
    09.06.2022
    Antigen
    H3K27ac
    Chargennummer
    6921014
    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. ABIN2667903 allows for H3K27Ac targeted digestion using CUT&RUN in mouse fore limb (11.5) cells.

    'Independent Validation' Siegel
    Validierungsbilder
    Protokoll
    Primärantikörper
    ABIN2667903
    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 (150,000 cells per sample).
      • Add 2 µL antibody (anti-H3K27ac antibody ABIN2667903, 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.
    Anmerkungen

    The protocol is published in PMID 36355069

    .
  • Konzentration
    1 μg/μL
    Buffer
    Purified IgG in PBS with 30 % glycerol and 0.035 % sodium azide.
    Konservierungsmittel
    Sodium azide
    Vorsichtsmaßnahmen
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Handhabung

    Avoid repeated freeze/thaw cycles and keep on ice when not in storage. This product is guaranteed for 6 months from date of receipt.

    Lagerung
    -20 °C
    Informationen zur Lagerung
    Antibodies in solution can be stored at -20°C for 2 years.
    Haltbarkeit
    6 months
  • Target
    Histone 3 (H3)
    Andere Bezeichnung
    Histone H3 (H3 Produkte)
    Synonyme
    H-3 antikoerper, histocompatibility 3 antikoerper, H3 antikoerper
    Molekulargewicht
    17 kDa
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