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Catalog No. ABIN3023251
  • Target See all Histone 3 (H3) Antibodies
    Histone 3 (H3)
    Binding Specificity
    • 60
    • 47
    • 45
    • 42
    • 42
    • 37
    • 36
    • 35
    • 35
    • 35
    • 34
    • 32
    • 31
    • 30
    • 29
    • 29
    • 26
    • 25
    • 23
    • 23
    • 22
    • 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
    H3K4me
    Reactivity
    • 1402
    • 941
    • 861
    • 59
    • 42
    • 41
    • 41
    • 40
    • 39
    • 35
    • 35
    • 30
    • 29
    • 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
    Host
    • 1219
    • 244
    • 8
    • 4
    Rabbit
    Clonality
    • 1120
    • 354
    • 1
    Polyclonal
    Conjugate
    • 824
    • 81
    • 63
    • 63
    • 63
    • 62
    • 62
    • 62
    • 37
    • 33
    • 23
    • 17
    • 17
    • 17
    • 17
    • 17
    • 14
    • 2
    • 1
    This Histone 3 antibody is un-conjugated
    Application
    • 1061
    • 411
    • 381
    • 316
    • 253
    • 188
    • 181
    • 179
    • 163
    • 161
    • 142
    • 126
    • 119
    • 48
    • 38
    • 34
    • 32
    • 20
    • 15
    • 14
    • 7
    • 7
    • 6
    • 4
    • 3
    • 2
    • 1
    • 1
    • 1
    Western Blotting (WB), Immunofluorescence (IF), Chromatin Immunoprecipitation (ChIP), Immunoprecipitation (IP), ChIP DNA-Sequencing (ChIP-seq), Cleavage Under Targets and Release Using Nuclease (CUT&RUN)
    Cross-Reactivity
    Human, Mouse, Rat
    Characteristics
    Methylated Antibodies
    Purification
    Affinity purification
    Immunogen
    A synthetic methylated peptide corresponding to residues surrounding K4 of human histone H3
    Isotype
    IgG
  • Application Notes
    WB 1:500 - 1:2000, IF 1:50 - 1:200, IP 1:50 - 1:200, ChIP 1:50 - 1:200, ChIP-seq 1:50 - 1:200, CUT&RUN 1:100
    Restrictions
    For Research Use only
  • Validation #104228 (Cleavage Under Targets and Release Using Nuclease)
    'Independent Validation' Badge
    by
    Mattias Pernebrink, Anna Nordin and Claudio Cantù; Cantù Lab, Gene Regulation during Development and Disease, Linköping University
    No.
    #104228
    Date
    11/12/2021
    Antigen
    H3K4me
    Lot Number
    3560036504
    Method validated
    Cleavage Under Targets and Release Using Nuclease
    Positive Control
    Recombinant anti-H3K27me3 CUT&RUN Positive Control antibody (antibodies-online, ABIN6923144)
    Negative Control
    Guinea Pig anti-rabbit IgG (antibodies-online, ABIN101961)
    Notes

    Passed. ABIN3023251 allows for H3K4me targeted digestion using CUT&RUN.

    'Independent Validation' Badge
    Validation Images
    Full Methods
    Primary Antibody
    ABIN3023251
    Secondary Antibody
    Full Protocol
    • Cell harvest
      • Harvest 250,000 HEK293T cells per antibody to be used at RT.
      • Centrifuge cell solution 3 min at 600 x g at RT.
      • Remove the liquid carefully.
      • Gently resuspend cells in 1 mL Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free) by pipetting and transfer cell solution to a 2 mL microcentrifuge tube.
      • Centrifuge cell solution 3 min at 600 x g at RT and discard the supernatant.
      • Repeat twice for a total of three washes.
      • Resuspend cell pellet in 1 mL Wash Buffer by gently pipetting.
    • Concanavalin A beads preparation
      • Prepare one 1.5 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.
    • Cell immobilization – binding to Concanavalin A beads
      • Carefully vortex the cell suspension and add 10 µL of the Con A beads in Binding Buffer to the cell suspension for each sample.
      • Close tube tightly and rotate for 10 min at RT.
    • Cell permeabilization and primary antibody binding
      • Divide cell suspension into separate 2 mL microcentrifuge tubes, one for each antibody (250,000 cells per sample).
      • Place the microcentrifuge tubes on a magnetic stand until the fluid is clear. Remove the liquid carefully.
      • Remove the microcentrifuge tubes from the magnetic stand.
      • Place each tube at a low angle on the vortex mixer set to a low speed and add 150 µL Digitonin Wash buffer (wash buffer with 0.025% (wt/vol) Digitonin) supplemented with 2 mM EDTA.
      • Gently vortex the microcentrifuge tubes until the beads are resuspended.
      • o Add 1.5 µL antibody (anti-H3K4me ABIN3023251, anti-H3K27me3 positive control antibody ABIN6923144, or guinea pig anti-rabbit IgG negative control antibody ABIN101961) to the respective tube, corresponding to a 1:100 dilution.
      • Rotate the microcentrifuge tubes ON at 4 °C.
      • Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
      • Remove the microcentrifuge tubes from the magnetic stand.
      • Resuspend with 1 mL Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 ml pipette tip.
      • Repeat once for a total of two washes.
    • pA-MNase Binding
      • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
      • Remove the microcentrifuge tubes from the magnetic stand.
      • Vortex the sample at low speed and add 150 μL CUTANA pAG-MNase 0.5X (antibodies-online ABIN6950951, 1:40 dilution of a 20X stock in Digitonin Wash Buffer) per sample, gently resuspending the beads by pipetting.
      • Rotate the microcentrifuge tubes for 1 h at 4 °C.
      • Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
      • Remove the microcentrifuge tubes from the magnetic stand.
      • Resuspend with 1 mL Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 mL pipette tip.
      • Repeat once for a total of two washes.
    • MNase digestion and release of pA-MNase-antibody-chromatin complexes
      • 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.
      • Place each tube at a low angle on the vortex mixer set to a low speed and add 100 μL Digitonin Wash buffer per sample along the side of the tube.
      • Place tubes in a heat block, kept on ice, and allow to chill.
      • Add 2 μL 0.1 M CaCl2 to each sample.
      • Incubate tubes at 0 °C for 15 min.
      • Add 100 μL 2X STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% (wt/vol) Digitonin, 100 μg/mL RNAse A, 50 μg/mL Glycogen).
      • Incubate tubes at 37 °C for 30 min.
      • Place the tubes on a magnet stand until the fluid is clear.
      • Transfer the supernatant containing the pAG-MNase-bound digested chromatin fragments to fresh 1.5 mL microcentrifuge tubes.
    • DNA extraction
      • Add 2 µL 10% SDS to a final concentration of 0.1% and 2.5 µL Proteinase K (20 mg/mL) to a final concentration of 0.25 mg/mL to each supernatant.
      • Gently vortex tubes at a low speed of approximately 1,100 rpm.
      • Incubate tubes at 50 °C for 1 h.
      • Add 200 µL PCI to tube.
      • Vortex tubes thoroughly at high speed until the liquid appears milky.
      • Centrifuge tubes in a tabletop centrifuge at 16,000 x g at RT for 5 min.
      • Carefully transfer to upper aqueous phase to a fresh 1.5 mL microcentrifuge tube containing 2 µL glycogen (diluted 1:10 to 2 mg/mL from the 20 mg/mL stock solution).
      • Add 20 µL 3 M NaOAc pH 5.2.
      • Add 400 µL 100% ethanol.
      • Place tubes for at -20 °C ON.
      • Centrifuge tubes in a tabletop centrifuge at 16,000 x g at 4 °C for 5min.
      • Remove the liquid carefully with a pipette.
      • Wash pellet with 1ml 70% ethanol.
      • Centrifuge tubes in a tabletop centrifuge at 16,000 x g at 4 °C for 1 min.
      • Remove the liquid carefully with a pipette.
      • Air-dry the pellet, then dissolve in 30 µL 1 mM Tris-HCl, 0.1 mM EDTA.
    • 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.
    • Peak calling
      • Trim reads using bbTools bbduk to remove adapters, artifacts and repeat sequences.
      • Aligned reads were mapped to the GRCh38 (hg38) human genome using bowtie2 with options --local --very-sensitive- local --no-unal --no-mixed --no-discordant - X 400.
      • Convert SAM files to BAM files and remove duplicates using SAMtools was used to convert SAM files to BAM files. Produce Bedgraph files with BEDtools genomecov.
      • Call peaks using SEACR against the IgG negative control with the options norm and relaxed.
    Experimental Notes

    The CUT&RUN alignment track was compared to a reference alignment track of ChIP-seq for H3K4me in HEK293 cells obtained from ENCODE (PMID 26527727), experiment ENCSR000FCG, track ENCFF274LAP.

  • Format
    Liquid
    Buffer
    PBS with 0.02 % sodium azide,50 % glycerol, pH 7.3.
    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 freeze / thaw cycles
    Storage
    -20 °C
    Storage Comment
    Store at -20°C. Avoid freeze / thaw cycles.
  • Zambanini, Nordin, Jonasson, Pagella, Cantù: "A new cut&run low volume-urea (LoV-U) protocol optimized for transcriptional co-factors uncovers Wnt/b-catenin tissue-specific genomic targets." in: Development (Cambridge, England), (2022) (PubMed).

    Zhang, Zhang, Cheng, Liu, Lin, Wu, Zhang, Wang, Wang, Guo, Zhang, Lei, Zhao, Zhu, Wan: "Functional characterization of rice CW-domain containing zinc finger proteins involved in histone recognition." in: Plant science : an international journal of experimental plant biology, Vol. 263, pp. 168-176, (2018) (PubMed).

    Chen, Zhang, Li, Feng, Zhang, Yao, Zhang, Wan: "Celastrol attenuates incision-induced inflammation and pain associated with inhibition of the NF-κB signalling pathway via SARM." in: Life sciences, Vol. 205, pp. 136-144, (2018) (PubMed).

    Cao, Liu, Yue, Liu, Pei, Gu, Wang, Jia: "Iron chelation inhibits cancer cell growth and modulates global histone methylation status in colorectal cancer." in: Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine, Vol. 31, Issue 5, pp. 797-805, (2018) (PubMed).

  • Target
    Histone 3 (H3)
    Alternative Name
    Histone H3 (H3 Products)
    Synonyms
    H-3 antibody, histocompatibility 3 antibody, H3 antibody
    Background
    Histones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. Nucleosomes consist of approximately 146 bp of DNA wrapped around a histone octamer composed of pairs of each of the four core histones (H2A, H2B, H3, and H4). The chromatin fiber is further compacted through the interaction of a linker histone, H1, with the DNA between the nucleosomes to form higher order chromatin structures. This gene is intronless and encodes a replication-dependent histone that is a member of the histone H3 family. Transcripts from this gene lack polyA tails, instead, they contain a palindromic termination element. This gene is located separately from the other H3 genes that are in the histone gene cluster on chromosome 6p22-p21.3.,H3.4,H3/g,H3FT,H3t,HIST3H3,Histone H3,HIST1H3A,Signal Transduction,MAPK-Erk Signaling Pathway,MAPK-P38 Signaling Pathway,Epigenetics & Nuclear Signaling,Epigenetic Modifications,Methylation,Histone H3
    Molecular Weight
    15 kDa
    Gene ID
    8290
    UniProt
    Q16695
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