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Histone 3 antibody (H3K36me3)

H3 Reactivity: Human, Mouse WB, ChIP, DB, ChIP-seq, CUT&RUN, CUT&Tag Host: Mouse Monoclonal MABI 0333 unconjugated
Catalog No. ABIN2668403
  • Target See all Histone 3 (H3) Antibodies
    Histone 3 (H3)
    Binding Specificity
    • 62
    • 47
    • 45
    • 43
    • 42
    • 37
    • 36
    • 35
    • 35
    • 35
    • 34
    • 32
    • 32
    • 31
    • 30
    • 29
    • 26
    • 25
    • 24
    • 23
    • 22
    • 22
    • 20
    • 18
    • 18
    • 17
    • 17
    • 16
    • 16
    • 16
    • 16
    • 16
    • 15
    • 14
    • 14
    • 13
    • 13
    • 12
    • 12
    • 12
    • 12
    • 12
    • 12
    • 11
    • 10
    • 10
    • 10
    • 10
    • 9
    • 8
    H3K36me3
    Reactivity
    • 1412
    • 941
    • 861
    • 59
    • 42
    • 41
    • 41
    • 40
    • 39
    • 35
    • 35
    • 30
    • 30
    • 22
    • 19
    • 6
    • 3
    • 3
    • 3
    • 3
    • 2
    • 2
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Human, Mouse
    Host
    • 1228
    • 245
    • 8
    • 5
    Mouse
    Clonality
    • 1129
    • 356
    • 1
    Monoclonal
    Conjugate
    • 828
    • 81
    • 63
    • 63
    • 63
    • 62
    • 62
    • 62
    • 37
    • 33
    • 23
    • 17
    • 17
    • 17
    • 17
    • 17
    • 14
    • 7
    • 2
    • 1
    This Histone 3 antibody is un-conjugated
    Application
    • 894
    • 392
    • 382
    • 319
    • 254
    • 188
    • 182
    • 182
    • 166
    • 160
    • 142
    • 126
    • 118
    • 48
    • 38
    • 35
    • 32
    • 20
    • 15
    • 13
    • 7
    • 7
    • 6
    • 4
    • 3
    • 2
    • 1
    • 1
    • 1
    Western Blotting (WB), Chromatin Immunoprecipitation (ChIP), Dot Blot (DB), ChIP DNA-Sequencing (ChIP-seq), Cleavage Under Targets and Release Using Nuclease (CUT&RUN), Cleavage Under Targets and Tagmentation (CUT&Tag)
    Purification
    Protein G Chromatography
    Immunogen
    This Histone H3 trimethylLys36 antibody was raised against a peptide containing trimethylLys36 of human Histone H3.
    Clone
    MABI 0333
    Isotype
    IgG1
  • Application Notes
    Recommended starting concentrations are
    ChIP: 5 - 10 µg per ChIP
    ChIP-Seq: 5 - 10 µg each
    WB: 0.5 - 2 µg/mL dilution
    DB: 0.5 - 2 µg/mL dilution
    CUT&RUN: 2 µL/200 µL reaction
    Optimal working dilution should be determined by the investigator.
    Restrictions
    For Research Use only
  • Validation #104510 (Cleavage Under Targets and Release Using Nuclease)
    'Independent Validation' Badge
    by
    Anna Nordin and Claudio Cantù; Cantù Lab, Gene Regulation during Development and Disease, Linköping University
    No.
    #104510
    Date
    08/14/2023
    Antigen
    H3K36me3
    Lot Number
    20822015
    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. ABIN2668403 allows for specific targeting of H3K36me3 in human cells using CUT&RUN.

    'Independent Validation' Badge
    Validation Images
    Full Methods
    Primary Antibody
    ABIN2668403
    Secondary Antibody
    Full Protocol
    • Cell harvest
      • Harvest 50,000 human fibroblast cells per antibody.
      • Centrifuge cell solution 3 min at 600 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 Buffer 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, ABIN6923139).
      • Pipette 10 µL Con A Beads slurry for each sample into the 1.5 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 each 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 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. 10 µL per sample.
    • Nuclei immobilization – binding to Concanavalin A beads
      • Carefully vortex the nuclei suspension and add 10 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 200µl of Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free) for each sample.
    • Cell permeabilization and primary antibody binding
      • Divide nuclei suspension into separate PCR tubes, one for each antibody (200 µL per sample).
      • Add 2 µL antibody (anti-H3K36me3 antibody ABIN2668403, 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 ON 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.
    • pAG-MNase Binding
      • Prepare a 1.5 mL microcentrifuge tube containing 200 µL of pAG mix for each sample (200 µl of wash buffer + 120 ng 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 200 µL of pAG-MNase premix.
      • Incubate for 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 for a total of five washes.
      • Resuspend in 200 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 51 µl of 2 mM CaCl2 mix per sample (50 µl Wash Buffer + 1 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 50 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 move the supernatant in fresh collection tubes with 3µl of EDTA/EGTA 0.25M (Digestion buffer).
      • Resuspend the sample in 47 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 1 h at 4 °C.
      • Transfer the supernatant containing the pAG-MNase-bound digested chromatin fragments to the previously collected digestion buffer.
    • 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), 36bp PE.
    • Bioinformatics
      • Align reads the human genome (hg38) using bowtie78 with settings -X 700 -m1 -v 3. Remove duplicate reads, and sort files using samtools. Filter mapped reads for size, keeping only reads with a fragment size at or below 120 base pairs.
      • Generate bedgraph files using bedtools genomecov.
      • Call peaks using SEACR version 1.3, in relaxed mode, normalizing to the negative control.
    Experimental Notes
  • Concentration
    0.44 μg/μL
    Buffer
    PBS pH 7.5 containing 30 % glycerol, 0.3 M NaCl, and 0.035 % sodium azide.
    Preservative
    Sodium azide
    Precaution of Use
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Handling Advice

    Avoid repeated freeze/thaw cycles and keep on ice when not in storage.

    Storage
    -20 °C
    Storage Comment
    Antibodies in solution can be stored at -20 °C for 2 years.
    Expiry Date
    6 months
  • Brahma, Henikoff: "RSC-Associated Subnucleosomes Define MNase-Sensitive Promoters in Yeast." in: Molecular cell, Vol. 73, Issue 2, pp. 238-249.e3, (2019) (PubMed).

    Powers, Parvanov, Baker, Walker, Petkov, Paigen: "The Meiotic Recombination Activator PRDM9 Trimethylates Both H3K36 and H3K4 at Recombination Hotspots In Vivo." in: PLoS genetics, Vol. 12, Issue 6, pp. e1006146, (2016) (PubMed).

    Xu, Gan, Zhou, Wee, Zhang, Ito: "Arabidopsis MRG domain proteins bridge two histone modifications to elevate expression of flowering genes." in: Nucleic acids research, Vol. 42, Issue 17, pp. 10960-74, (2014) (PubMed).

    Rechtsteiner, Ercan, Takasaki, Phippen, Egelhofer, Wang, Kimura, Lieb, Strome: "The histone H3K36 methyltransferase MES-4 acts epigenetically to transmit the memory of germline gene expression to progeny." in: PLoS genetics, Vol. 6, Issue 9, pp. e1001091, (2010) (PubMed).

  • Target
    Histone 3 (H3)
    Alternative Name
    Histone H3 (H3 Products)
    Synonyms
    H-3 antibody, histocompatibility 3 antibody, H3 antibody
    Molecular Weight
    17 kDa
    Gene ID
    3020
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