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Epigenetics and Nuclear Signaling Histones H3 Methylated
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RecombinantRabMAb

Recombinant Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

  • Datasheet
Reviews (10)Q&A (3)References (142)

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ChIP-sequencing - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
  • ChIP - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
  • ChIP-sequencing - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
  • Immunoprecipitation - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
  • Flow Cytometry - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
  • Western blot - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
  • Flow Cytometry - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
  • Western blot - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
  • Flow Cytometry - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
  • Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [Y47] to Histone H3 (di methyl K4) - ChIP Grade
  • Suitable for: ChIP, ChIP-sequencing, WB, IHC-P, ICC/IF, IP, Flow Cyt
  • Reacts with: Mouse, Rat, Chicken, Cow, Human, African green monkey

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Overview

  • Product name

    Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade
    See all Histone H3 primary antibodies
  • Description

    Rabbit monoclonal [Y47] to Histone H3 (di methyl K4) - ChIP Grade
  • Host species

    Rabbit
  • Specificity

    This antibody only detects Histone H3 dimethylated on Lysine 4.
  • Tested Applications & Species

    Application Species
    ChIP
    Human
    ChIP-seq
    Human
    Flow Cyt
    Human
    ICC/IF
    Human
    IHC-P
    Mouse
    Rat
    Human
    IP
    Human
    WB
    Rat
    Human
    See all applications and species data
  • Immunogen

    Synthetic peptide within Human Histone H3 aa 1-100 (di methyl K4). The exact sequence is proprietary.

  • Positive control

    • WB: HeLa, HEK-293, SH-SY5Y, C6 L6, NIH/3T3, COS-1, UMNSAH/DF-1 and MDBK cell lysates. ICC/IF: HepG2 cells. Flow Cyt: HeLa and HepG2 cells. IHC-P: Human cervical carcinoma, mouse colon and rat spleen tissues. ChIP: Chromatin prepared from Hela cells, ALDO ChIP primer pair ab269260. ChIP-seq: Chromatin prepared from Hela cells. IP: Hep-G2 cells.
  • General notes

    Every new batch of this antibody is tested in house in ChIP.

     

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    Y47
  • Isotype

    IgG
  • Research areas

    • Epigenetics and Nuclear Signaling
    • Histones
    • H3
    • Methylated
    • Epigenetics and Nuclear Signaling
    • ChIP assays
    • ChIP antibodies

Associated products

  • Alternative Versions

    • Anti-Histone H3 (di methyl K4) antibody [Y47] - BSA and Azide free (ab173324)
    • Alexa Fluor® 488 Anti-Histone H3 (di methyl K4) antibody [Y47] (ab200024)
    • Alexa Fluor® 647 Anti-Histone H3 (di methyl K4) antibody [Y47] (ab200026)
    • Alexa Fluor® 594 Anti-Histone H3 (di methyl K4) antibody [Y47] (ab207864)
    • Alexa Fluor® 405 Anti-Histone H3 (di methyl K4) antibody [Y47] (ab207865)
    • PE Anti-Histone H3 (di methyl K4) antibody [Y47] (ab208741)
  • ChIP Related Products

    • Rabbit Anti-Mouse IgG H&L (ab46540)
    • ChIP Kit (ab500)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (ab96899)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Positive Controls

    • HeLa nuclear extract lysate (ab14655)
    • ALDOA positive control ChIP primer pair (ab269260)
    • HeLa whole cell lysate (ab29545)
  • Recombinant Protein

    • Recombinant human Histone H3 protein (Active) (ab198757)
  • Related Products

    • Histone H3 (K4) Methyltransferase Activity Quantification Assay Kit (ab113452)
    • Histone Demethylase (H3K4) Activity Quantification Assay Kit (ab113455)
    • Histone H3 (di-methyl K4) Quantification Kit (Colorimetric) (ab115052)
    • Prestained Protein Ladder - Broad molecular weight (10-245 kDa) (ab116028)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab32356 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

Application Species
ChIP
Human
ChIP-seq
Human
Flow Cyt
Human
ICC/IF
Human
IHC-P
Mouse
Rat
Human
IP
Human
WB
Rat
Human
All applications
Sheep
Saccharomyces cerevisiae
Xenopus laevis
Arabidopsis thaliana
Monkey
Zebrafish
Mammals
Rice
Application Abreviews Notes
ChIP (1)
Use 2 µg for 25 µg of chromatin.

Use ALDOA ChIP primer pair ab269260 as positive control.

ChIP-sequencing
Use 4 µg for 30 µg of chromatin.
WB (2)
1/2000. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).
IHC-P (1)
1/800. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ICC/IF (3)
1/1000.
IP
1/30.
Flow Cyt
1/20 - 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Notes
ChIP
Use 2 µg for 25 µg of chromatin.

Use ALDOA ChIP primer pair ab269260 as positive control.

ChIP-sequencing
Use 4 µg for 30 µg of chromatin.
WB
1/2000. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).
IHC-P
1/800. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ICC/IF
1/1000.
IP
1/30.
Flow Cyt
1/20 - 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Target

  • Function

    Core component of nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling.
  • Sequence similarities

    Belongs to the histone H3 family.
  • Developmental stage

    Expressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation.
  • Post-translational
    modifications

    Acetylation is generally linked to gene activation. Acetylation on Lys-10 (H3K9ac) impairs methylation at Arg-9 (H3R8me2s). Acetylation on Lys-19 (H3K18ac) and Lys-24 (H3K24ac) favors methylation at Arg-18 (H3R17me).
    Citrullination at Arg-9 (H3R8ci) and/or Arg-18 (H3R17ci) by PADI4 impairs methylation and represses transcription.
    Asymmetric dimethylation at Arg-18 (H3R17me2a) by CARM1 is linked to gene activation. Symmetric dimethylation at Arg-9 (H3R8me2s) by PRMT5 is linked to gene repression. Asymmetric dimethylation at Arg-3 (H3R2me2a) by PRMT6 is linked to gene repression and is mutually exclusive with H3 Lys-5 methylation (H3K4me2 and H3K4me3). H3R2me2a is present at the 3' of genes regardless of their transcription state and is enriched on inactive promoters, while it is absent on active promoters.
    Methylation at Lys-5 (H3K4me), Lys-37 (H3K36me) and Lys-80 (H3K79me) are linked to gene activation. Methylation at Lys-5 (H3K4me) facilitates subsequent acetylation of H3 and H4. Methylation at Lys-80 (H3K79me) is associated with DNA double-strand break (DSB) responses and is a specific target for TP53BP1. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me) are linked to gene repression. Methylation at Lys-10 (H3K9me) is a specific target for HP1 proteins (CBX1, CBX3 and CBX5) and prevents subsequent phosphorylation at Ser-11 (H3S10ph) and acetylation of H3 and H4. Methylation at Lys-5 (H3K4me) and Lys-80 (H3K79me) require preliminary monoubiquitination of H2B at 'Lys-120'. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me) are enriched in inactive X chromosome chromatin.
    Phosphorylated at Thr-4 (H3T3ph) by GSG2/haspin during prophase and dephosphorylated during anaphase. Phosphorylation at Ser-11 (H3S10ph) by AURKB is crucial for chromosome condensation and cell-cycle progression during mitosis and meiosis. In addition phosphorylation at Ser-11 (H3S10ph) by RPS6KA4 and RPS6KA5 is important during interphase because it enables the transcription of genes following external stimulation, like mitogens, stress, growth factors or UV irradiation and result in the activation of genes, such as c-fos and c-jun. Phosphorylation at Ser-11 (H3S10ph), which is linked to gene activation, prevents methylation at Lys-10 (H3K9me) but facilitates acetylation of H3 and H4. Phosphorylation at Ser-11 (H3S10ph) by AURKB mediates the dissociation of HP1 proteins (CBX1, CBX3 and CBX5) from heterochromatin. Phosphorylation at Ser-11 (H3S10ph) is also an essential regulatory mechanism for neoplastic cell transformation. Phosphorylated at Ser-29 (H3S28ph) by MLTK isoform 1, RPS6KA5 or AURKB during mitosis or upon ultraviolet B irradiation. Phosphorylation at Thr-7 (H3T6ph) by PRKCBB is a specific tag for epigenetic transcriptional activation that prevents demethylation of Lys-5 (H3K4me) by LSD1/KDM1A. At centromeres, specifically phosphorylated at Thr-12 (H3T11ph) from prophase to early anaphase, by DAPK3 and PKN1. Phosphorylation at Thr-12 (H3T11ph) by PKN1 is a specific tag for epigenetic transcriptional activation that promotes demethylation of Lys-10 (H3K9me) by KDM4C/JMJD2C. Phosphorylation at Tyr-42 (H3Y41ph) by JAK2 promotes exclusion of CBX5 (HP1 alpha) from chromatin.
    Monoubiquitinated by RAG1 in lymphoid cells, monoubiquitination is required for V(D)J recombination (By similarity). Ubiquitinated by the CUL4-DDB-RBX1 complex in response to ultraviolet irradiation. This may weaken the interaction between histones and DNA and facilitate DNA accessibility to repair proteins.
  • Cellular localization

    Nucleus. Chromosome.
  • Target information above from: UniProt accession P68431 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 8350 Human
    • Entrez Gene: 8351 Human
    • Entrez Gene: 8352 Human
    • Entrez Gene: 8353 Human
    • Entrez Gene: 8354 Human
    • Entrez Gene: 8355 Human
    • Entrez Gene: 8356 Human
    • Entrez Gene: 8357 Human
    • Entrez Gene: 8358 Human
    • Entrez Gene: 8968 Human
    • Entrez Gene: 319152 Mouse
    • Entrez Gene: 319153 Mouse
    • Entrez Gene: 360198 Mouse
    • Entrez Gene: 97908 Mouse
    • Entrez Gene: 100364501 Rat
    • Entrez Gene: 100365669 Rat
    • Entrez Gene: 291159 Rat
    • Entrez Gene: 679994 Rat
    • Entrez Gene: 680511 Rat
    • Entrez Gene: 682330 Rat
    • Omim: 602810 Human
    • SwissProt: P68432 Cow
    • SwissProt: P68431 Human
    • SwissProt: P68433 Mouse
    • SwissProt: Q6LED0 Rat
    • Unigene: 132854 Human
    • Unigene: 247813 Human
    • Unigene: 247814 Human
    • Unigene: 248176 Human
    • Unigene: 443021 Human
    • Unigene: 484990 Human
    • Unigene: 532144 Human
    • Unigene: 533292 Human
    • Unigene: 546315 Human
    • Unigene: 586261 Human
    • Unigene: 591778 Human
    • Unigene: 221301 Mouse
    • Unigene: 261657 Mouse
    • Unigene: 377874 Mouse
    • Unigene: 390558 Mouse
    • Unigene: 397328 Mouse
    • Unigene: 138090 Rat
    see all
  • Alternative names

    • H3 histone family member E pseudogene antibody
    • H3 histone family, member A antibody
    • H3/A antibody
    • H31_HUMAN antibody
    • H3F3 antibody
    • H3FA antibody
    • Hist1h3a antibody
    • HIST1H3B antibody
    • HIST1H3C antibody
    • HIST1H3D antibody
    • HIST1H3E antibody
    • HIST1H3F antibody
    • HIST1H3G antibody
    • HIST1H3H antibody
    • HIST1H3I antibody
    • HIST1H3J antibody
    • HIST3H3 antibody
    • histone 1, H3a antibody
    • Histone cluster 1, H3a antibody
    • Histone H3 3 pseudogene antibody
    • Histone H3.1 antibody
    • Histone H3/a antibody
    • Histone H3/b antibody
    • Histone H3/c antibody
    • Histone H3/d antibody
    • Histone H3/f antibody
    • Histone H3/h antibody
    • Histone H3/i antibody
    • Histone H3/j antibody
    • Histone H3/k antibody
    • Histone H3/l antibody
    see all

Images

  • ChIP-sequencing - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
    ChIP-sequencing - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

    Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 107 cells and 4 µg of Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356). ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.

     Additional screenshots of mapped reads can be downloaded here.

  • ChIP - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
    ChIP - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

    Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab32356 (red), and 20µl of Anti Rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach). Primers and probes are located in the first kb of the transcribed region.

  • ChIP-sequencing - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
    ChIP-sequencing - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

    Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 30 µg of chromatin and 4 µg of Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356). ChIP DNA was sequenced on the Illumina NextSeq 500 to a depth of 30 million reads. ChIP-Seq validation performed by Active Motif, Carlsbad, CA.

     Additional screenshots of mapped reads can be downloaded here.

  • Immunoprecipitation - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
    Immunoprecipitation - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

    ab32356 (purified) at 1/30 dilution (20 µg/ml) immunoprecipitating Histone H3 di methyl K4 in HepG2 whole cell lysate.

    Lane 1 (input): HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate 10µg
    Lane 2 (+): ab32356 & HepG2 whole cell lysate
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32356 in HepG2 whole cell lysate
    For western blotting, ab32356 at 1/500 and veriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/1000 dilution.

    Blocking and diluting buffer: 5% NFDM /TBST.

  • Flow Cytometry - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
    Flow Cytometry - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

    Flow Cytometry analysis of HepG2 (human hepatocellular carcinomal) cells labeling Histone H3 di methyl K4 with purified ab32356 at 1:500 dilution (1.17 µg/ml) (Red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) / Black. Unlabeled control - Unlabelled cells / blue.

  • Western blot - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
    Western blot - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
    All lanes : Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356) at 1/10000 dilution (purified)

    Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
    Lane 2 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
    Lane 3 : SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate
    Lane 4 : C6 (Rat glial tumor cell line) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

    Predicted band size: 15 kDa
    Additional bands at: 17 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 5 seconds
  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
    Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

    Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling Histone H3 (di methyl K4) with purified ab32356 at a dilution of 1/1000. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin (1/1000) using ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary antibody. Nuclei counterstained with DAPI (blue). 

    Control 1: primary antibody (1/1000) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000).

    Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000).

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling Histone H3 (di methyl K4) with purified ab32356 at a dilution of 1/800. Antigen retrieval was performed using Tris/EDTA buffer, pH9. ab97051, a HRP-conjugated goat anti-rabbit IgG H&L was used as the secondary antibody (1/500). Counter stained with hematoxylin.

  • Flow Cytometry - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
    Flow Cytometry - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

    Flow Cytometry analysis of HeLa cells labelling Histone H3 (di methyl K4) with purified ab32356 at 1/150 (red). Cells were fixed with 80% methanol. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG (ab172730). Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

  • Western blot - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
    Western blot - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
    All lanes : Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356) at 1/2000 dilution (purified)

    Lane 1 : L6 (Rat skeletal muscle cell line) whole cell lysate
    Lane 2 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
    Lane 3 : COS-1 (African green monkey kidney fibroblast-like cell line) whole cell lysate
    Lane 4 : UMNSAH/DF-1 (Transformed chicken embryonic fibroblast cell line) whole cell lysate
    Lane 5 : MDBK (Bovine kidney cell line) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

    Predicted band size: 15 kDa
    Observed band size: 17 kDa
    why is the actual band size different from the predicted?


    Exposure time: 5 seconds
  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
    Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

    ICC/IF image of unpurified ab32356 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32356 , 5µg/ml) overnight at +4°C. The secondary antibody (green) was a goat anti-rabbit DyLight® 488 (IgG - H&L, pre-adsorbed) (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse colon tissue labelling Histone H3 (di methyl K4) with purified ab32356 at a dilution of 1/800. Antigen retrieval was performed using Tris/EDTA buffer, pH9. ab97051, a HRP-conjugated goat anti-rabbit IgG H&L was used as the secondary antibody (1/500). Counter stained with hematoxylin.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat spleen tissue labelling Histone H3 (di methyl K4) with purified ab32356 at a dilution of 1/800. Antigen retrieval was performed using Tris/EDTA buffer, pH9. ab97051, a HRP-conjugated goat anti-rabbit IgG H&L was used as the secondary antibody (1/500). Counter stained with hematoxylin.

  • Flow Cytometry - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
    Flow Cytometry - Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

    Overlay histogram showing HeLa cells stained with unpurified ab32356 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32356, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was a goat anti-rabbit DyLight® 488 (IgG; H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)
    Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade (ab32356)

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

Datasheets and documents

    • Datasheet
  • References (142)

    Publishing research using ab32356? Please let us know so that we can cite the reference in this datasheet.

    ab32356 has been referenced in 142 publications.

    • Toska E  et al. PI3K Inhibition Activates SGK1 via a Feedback Loop to Promote Chromatin-Based Regulation of ER-Dependent Gene Expression. Cell Rep 27:294-306.e5 (2019). PubMed: 30943409
    • Ishihara H  et al. Primed histone demethylation regulates shoot regenerative competency. Nat Commun 10:1786 (2019). PubMed: 30992430
    • Yin X  et al. Histone demethylase RBP2 mediates the blast crisis of chronic myeloid leukemia through an RBP2/PTEN/BCR-ABL cascade. Cell Signal 63:109360 (2019). PubMed: 31374292
    • Tajima K  et al. SETD1A protects from senescence through regulation of the mitotic gene expression program. Nat Commun 10:2854 (2019). PubMed: 31253781
    • Zhang W  et al. Knockdown of KDM1A suppresses tumour migration and invasion by epigenetically regulating the TIMP1/MMP9 pathway in papillary thyroid cancer. J Cell Mol Med 23:4933-4944 (2019). PubMed: 31211500
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-10 of 13 Abreviews or Q&A

    Immunocytochemistry/ Immunofluorescence abreview for Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Mouse Cell (NIH3T3)
    Specification
    NIH3T3
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 0.1% Triton
    Blocking step
    Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 24°C
    Read More

    Abcam user community

    Verified customer

    Submitted Mar 22 2013

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (Colon)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: EDTA
    Permeabilization
    Yes - PBS-Tween
    Specification
    Colon
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
    Fixative
    Formaldehyde
    Read More

    Abcam user community

    Verified customer

    Submitted Feb 03 2021

    Western blot abreview for Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Fruit fly (Drosophila melanogaster) Tissue lysate - whole (Whole fly tissue)
    Gel Running Conditions
    Reduced Denaturing (gradient precasted gel, Biorad 567-8124)
    Loading amount
    40 µg
    Specification
    Whole fly tissue
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted May 06 2016

    Question

    Is this antibody specific to Histone H3 (di methyl K4)?

    Read More

    Abcam community

    Verified customer

    Asked on Dec 10 2012

    Answer


    For ab32356 (https://www.abcam.com/histone-h3-di-methyl-k4-antibody-y47-chip-grade-ab32356.html):
    Based on our western blot testing testing, ab32356 should specifically detect Histone H3 dimethylated on Lysine 4. The WB figure on the product datasheet shows that no signal was observed on non-methylated recombinant Histone H3 (˜1ug).
    Recommended amount: Use 8ul for 25ug of chromatin

    For ab7766 (https://www.abcam.com/histone-h3-di-methyl-k4-antibody-chip-grade-ab7766.html):
    All batches of ab7766 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - di methyl K4 peptide (ab7768), indicating that this antibody specifically recognizes the Histone H3 - di methyl K4 modification. I've attached the results of one of these peptide arrays to this email.
    Recommended amount: Use 2ug for 25ug of chromatin

    Read More

    Abcam Scientific Support

    Answered on Dec 10 2012

    Immunohistochemistry (Frozen sections) abreview for Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Rat Tissue sections (Rat airway epithelium and alveoli)
    Specification
    Rat airway epithelium and alveoli
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 0.1% Triton
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 27°C
    Read More

    Abcam user community

    Verified customer

    Submitted Nov 15 2012

    Immunohistochemistry (Frozen sections) abreview for Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Human Tissue sections (Human lung endothelial lining)
    Specification
    Human lung endothelial lining
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 0.1% Triton
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 27°C
    Read More

    Abcam user community

    Verified customer

    Submitted Nov 15 2012

    Immunohistochemistry (Frozen sections) abreview for Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Mouse Tissue sections (Mouse bronchiolar epithelium)
    Specification
    Mouse bronchiolar epithelium
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 0.1% Triton
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 27°C
    Read More

    Abcam user community

    Verified customer

    Submitted Nov 15 2012

    Immunocytochemistry/ Immunofluorescence abreview for Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Monkey Cell (COS1)
    Specification
    COS1
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 0.1% Triton X-100
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted Nov 06 2012

    Western blot abreview for Anti-Histone H3 (di methyl K4) antibody [Y47] - ChIP Grade

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Monkey Cell lysate - whole cell (COS1)
    Loading amount
    20 µg
    Specification
    COS1
    Gel Running Conditions
    Reduced Denaturing (12%)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted Nov 05 2012

    Question

    The concentration seems very low though – I have eight other antibodies at concentrations of 0.6 – 1 mg/ml. Is 0.04 mg/ml the usual concentration for this antibody?  

    Read More

    Abcam community

    Verified customer

    Asked on Feb 28 2012

    Answer

    While the IgG produced by rabbits have greater affinity and greater sensitivity than mouse IgG (about 100-1000 fold), the total amount of IgG produced by the rabbit hybridomas is generally lower than mice. Since many people are not aware of this fact providing the antibody concentration may be misleading.

    Many veteran antibody users are generally familiar with the IgG amount provided from mice antibodies and can judge how many reactions they may get or need to use based on concentration. This experience is not transferable to rabbit mAbs, the concentration scales are generally very different. Since the specificity and sensitivity is much higher with rabbit mAbs, less IgG is required per reaction.

    The important thing to focus on is that while the amount of IgG may not be comparable to mouse mAbs, customers are receiving a comparable number of reactions/tests/blots/stainings per tube. Price per reaction is very comparable and competitive.

    Read More

    Abcam Scientific Support

    Answered on Feb 28 2012

    1-10 of 13 Abreviews or Q&A

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