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  1. Link

    histone-h3-mono-methyl-k9-antibody-chip-grade-ab9045.pdf

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Epigenetics and Nuclear Signaling Histones H3 Methylated
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Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)

  • Datasheet
  • SDS
Reviews (18)Q&A (2)References (99)

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ChIP - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)
  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)
  • Western blot - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)
  • Western blot - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)
  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)
  • Immunoprecipitation - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)

Key features and details

  • Rabbit polyclonal to Histone H3 (mono methyl K9) - ChIP Grade
  • Suitable for: IP, WB, ChIP, ICC/IF
  • Reacts with: Cow, Human, Arabidopsis thaliana
  • Isotype: IgG

You may also be interested in

Protein
Product image
Recombinant human Histone H3 protein (Active) (ab198757)
Secondary
Product image
Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
Primary
Product image
Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - BSA and Azide free (ab222481)

View more associated products

Overview

  • Product name

    Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade
    See all Histone H3 primary antibodies
  • Description

    Rabbit polyclonal to Histone H3 (mono methyl K9) - ChIP Grade
  • Host species

    Rabbit
  • Specificity

    Weak cross reactivity is observed with mono methyl K27 Histone H3. No cross-reactivity is seen with di or tri methyl K27.
  • Tested applications

    Suitable for: IP, WB, ChIP, ICC/IFmore details
  • Species reactivity

    Reacts with: Cow, Human, Arabidopsis thaliana
    Predicted to work with: Mouse, Rat, Saccharomyces cerevisiae, Xenopus laevis, Indian muntjac, Schizosaccharomyces pombe, Mammals
  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
    (Peptide available as ab1771)

  • General notes

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

    If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

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 or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.40
    Preservative: 0.02% Sodium azide
    Constituent: PBS

    Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
  • Concentration information loading...
  • Purity

    Immunogen affinity purified
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Research areas

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

Associated products

  • 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)
  • Control Peptide

    • Human Histone H3 (mono methyl K4) peptide (ab1340)
    • Human Histone H3 (tri methyl K4) peptide (ab1342)
    • Human Histone H3 (di methyl K9) peptide (ab1772)
    • Human Histone H3 (tri methyl K9) peptide (ab1773)
    • Human Histone H3 (mono methyl K27) peptide (ab1780)
    • Human Histone H3 (tri methyl K27) peptide (ab1782)
    • Human Histone H3 (unmodified) peptide (ab7228)
    • Human Histone H3 (di methyl K4) peptide (ab7768)
  • Immunizing Peptide (Blocking)

    • Human Histone H3 (mono methyl K9) peptide (ab1771)
  • Isotype control

    • Rabbit IgG, polyclonal - Isotype Control (ChIP Grade) (ab171870)
  • Recombinant Protein

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

    • Histone H3 (K9) Methyltransferase Activity Quantification Assay Kit (ab113453)
    • Histone H3 (mono-methyl K9) Quantification Kit (Colorimetric) (ab115060)
    • Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab9045 in the following tested applications.

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

Application Abreviews Notes
IP
Use at an assay dependent concentration.
WB (7)
1/1000. Detects a band of approximately 15 kDa (predicted molecular weight: 17 kDa). Can be blocked with Histone H3 peptide - mono methyl K9 (ab1771).
ChIP (1)
Use 4-5µg for 106 cells.
ICC/IF (4)
Use at an assay dependent concentration.
Notes
IP
Use at an assay dependent concentration.
WB
1/1000. Detects a band of approximately 15 kDa (predicted molecular weight: 17 kDa). Can be blocked with Histone H3 peptide - mono methyl K9 (ab1771).
ChIP
Use 4-5µg for 106 cells.
ICC/IF
Use at an assay dependent concentration.

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 - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)
    ChIP - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)

    Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 µg of chromatin, 2 µg of ab9045 (blue), and 20 µl of protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)
    Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)This image is courtesy of Kirk McManus in the lab of Michael Hendzel, Univeristy of Alberta

    Anti-mono methyl lysine 9 of histone H3 (green) has a distribution often associated with euchromatic probes (small foci).  Most of these foci localize to regions that contain obvious enrichments of DNA with DAPI staining (red).  The perinucleolar chromatin is typically a site enriched in monomethylated lysine 9. 

    Top left: Mono-methyl Lys 9 (ab9045); Bottom left: DAPI; Top right: Merge of ab9045 (green) and DAPI (red).

  • Western blot - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)
    Western blot - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)
    All lanes : Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045) at 1 µg/ml

    Lane 2 : Human Histone H3 (unmodified) peptide (ab7228)
    Lane 3 : Human Histone H3 (mono methyl K27) peptide (ab1780)
    Lane 4 : Human Histone H3 (di methyl K27) peptide (ab1781)
    Lane 5 : Human Histone H3 (tri methyl K27) peptide (ab1782)
    Lane 6 : Human Histone H3 (mono methyl K4) peptide (ab1340)
    Lane 7 : Human Histone H3 (mono methyl K9) peptide (ab1771)

    Predicted band size: 17 kDa



    Rabbit polyclonal to Histone H3 K9 Methyl K9 (1/1000)

    Peptides at 1 ug/ml

    1XTBS, 5%BSA, 0.5% Tween

    This antibody shows significantly greater reactivity with mono methyl K9. This can be seen in lane 7, as the addition of ab1771 (mono methyl K9) completely blocks the activity of ab9045. Weaker cross-reactivity is seen against mono methyl K27. This is shown in lane 3, as the addition of ab1780 only partially blocks the activity of ab9045.

  • Western blot - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)
    Western blot - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)
    All lanes : Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045) at 1 µg/ml

    Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate (ab121)
    Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (unmodified) peptide (ab7228) at 0.5 µg/ml
    Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.5 µg/ml
    Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (di methyl K4) peptide (ab7768) at 0.5 µg/ml
    Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (tri methyl K4) peptide (ab1342) at 0.5 µg/ml
    Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.5 µg/ml
    Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (di methyl K9) peptide (ab1772) at 0.5 µg/ml
    Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (tri methyl K9) peptide (ab1773) at 0.5 µg/ml
    Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (mono methyl K27) peptide (ab1780) at 0.5 µg/ml
    Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (di methyl K27) peptide (ab1781) at 0.5 µg/ml
    Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (tri methyl K27) peptide (ab1782) at 0.5 µg/ml

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 17 kDa

  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)
    Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)

    ab9045 staining Histone H3 (mono methyl K9) in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Triton for 1h. The cells were then incubated with the antibody ab9045 at 0.1µg/ml and ab7291 (Mouse monoclonal to alpha Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

  • Immunoprecipitation - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)
    Immunoprecipitation - Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade (ab9045)

    Histone H3 (mono methyl K9) was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Histone H3 (mono methyl K9) and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab9045.
    Secondary: Anti-rabbit IgG VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution.
    Band: 17kDa: Histone H3 (mono methyl K9).

Protocols

  • Chromatin Immunofluorescence

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (99)

Publishing research using ab9045? Please let us know so that we can cite the reference in this datasheet.

ab9045 has been referenced in 99 publications.

  • Sak A  et al. Chaetocin induced chromatin condensation: effect on DNA repair signaling and survival. Int J Radiat Biol 97:494-506 (2021). PubMed: 33428851
  • Bukova I  et al. Loss of Wiz Function Affects Methylation Pattern in Palate Development and Leads to Cleft Palate. Front Cell Dev Biol 9:620692 (2021). PubMed: 34150743
  • Neupert J  et al. An epigenetic gene silencing pathway selectively acting on transgenic DNA in the green alga Chlamydomonas. Nat Commun 11:6269 (2020). PubMed: 33293544
  • Tang DE  et al. Histone Demethylase JMJD1A Promotes Tumor Progression via Activating Snail in Prostate Cancer. Mol Cancer Res 18:698-708 (2020). PubMed: 32019811
  • Rowbotham K  et al. Differential SP1 interactions in SV40 chromatin from virions and minichromosomes. Virology 548:124-131 (2020). PubMed: 32838933
View all Publications for this product

Customer reviews and Q&As

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1-10 of 20 Abreviews or Q&A

Western blot abreview for Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade

Excellent
Abreviews
Abreviews
abreview image
Application
Western blot
Sample
Solanum lycopersicum (Tomato) Tissue lysate - nuclear (histone extraction)
Gel Running Conditions
Reduced Denaturing (12% SDS PAGE)
Loading amount
5 µg
Specification
histone extraction
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Jose Manuel

Verified customer

Submitted Jan 24 2020

Western blot abreview for Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade

Average
Abreviews
Abreviews
abreview image
Application
Western blot
Sample
Saccharomyces cerevisiae Cell lysate - whole cell (Whole cell lysate)
Gel Running Conditions
Reduced Denaturing (15%)
Loading amount
60000 cells
Specification
Whole cell lysate
Blocking step
Odyssey blocking buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 50% · Temperature: 25°C
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Aug 08 2016

Immunohistochemistry (Frozen sections) abreview for Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade

Excellent
Abreviews
Abreviews
abreview image
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Skin)
Permeabilization
No
Specification
Skin
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 22°C
Fixative
Paraformaldehyde
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Ahmar Aziz

Verified customer

Submitted Jul 08 2016

Immunocytochemistry/ Immunofluorescence abreview for Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade

Excellent
Abreviews
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (Brain cultured cells)
Specification
Brain cultured cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Oct 12 2015

Immunohistochemistry (PFA perfusion fixed frozen sections) abreview for Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade

Excellent
Abreviews
Abreviews
abreview image
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Mouse Tissue sections (Brain)
Permeabilization
Yes - Triton x-100, 0.01%
Specification
Brain
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Jun 18 2015

Immunohistochemistry (Frozen sections) abreview for Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade

Excellent
Abreviews
Abreviews
abreview image
Application
Immunohistochemistry (Frozen sections)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Sample
Mouse Tissue sections (Brain)
Specification
Brain
Permeabilization
Yes - Triton x-100, 0.01%
Fixative
Paraformaldehyde
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The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

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Verified customer

Submitted Mar 30 2015

Western blot abreview for Anti-Histone H3 (mono methyl K9) antibody - ChIP Grade

Average
Abreviews
Abreviews
abreview image
Application
Western blot
Loading amount
40 µg
Gel Running Conditions
Reduced Denaturing (10% PAGE)
Sample
Mouse Cell lysate - whole cell (Cardiomyocytes)
Specification
Cardiomyocytes
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

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Verified customer

Submitted Jan 30 2015

Immunocytochemistry/ Immunofluorescence abreview for Anti-Histone H3 (methylated) antibody - ChIP Grade

Average
Abreviews
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Cyanidioschyzon merolae Cell (Synchronous culture using light/dark cycle)
Specification
Synchronous culture using light/dark cycle
Fixative
Paraformaldehyde
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 37°C
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The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

俊之 曾根

Verified customer

Submitted Feb 12 2013

Western blot abreview for Anti-Histone H3 (methylated) antibody - ChIP Grade

Excellent
Abreviews
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - nuclear (rhabdomyosarcoma cell lines)
Loading amount
25 µg
Specification
rhabdomyosarcoma cell lines
Gel Running Conditions
Reduced Denaturing (10-20% tris glycine)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
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The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Jan 30 2013

Question

Customer inquiry regarding anti-histone H3 antibodies. Study is investigating methylation status of Histone H3, specifically on Lysine 9 (K9). Products I am already considering are ab70550 (anti-histone H3), ab1220 (anti-histone H3 di methyl K9), ab8898 (anti-histone H3 tri methyl K9), ab3790 (anti-KMT8/Riz1/Riz2). What protocol do you recommend in regards to preparing a cell lysate for detecting histone H3 via western blot? What is the minimum amount of cells that should be used? Why would one investigate these antibodies together in one study?

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Abcam community

Verified customer

Asked on Nov 21 2012

Answer

Below I have summarized our conversation and answers to your questions.

For detecting nuclear proteins via western blot we recommend using a RIPA buffer or cell fractionation protocol in preparation of your cell lysate. I attached our protocol book to this email for information regarding specific cell lysate preparation procedures. Below are the fractionation kits that we talked about during your phone call:
Nuclear Extraction Kit (ab113474) https://www.abcam.com/episeeker-nuclear-extraction-kit-ab113474.html
Histone Extraction Kit (ab113476)
https://www.abcam.com/episeeker-histone-extraction-kit-ab113476.html

The Histone Extraction Kit can be used to extract histones from as low as 1 million cells or 1 mg of tissue as stated on the datasheet. These lower limits are good guidelines for the Nuclear Extraction Kit as well.

Since ab1220 (anti-histone H3 di methyl K9) is the only antibody in your provided list that is raised in mouse (the others are raised in rabbit), we discussed substituting the rabbit monoclonal antibody ab32521 (https://www.abcam.com/histone-h3-di-methyl-k9-antibody-y49-ab32521.html) (anti-histone H3 di methyl K9) so you can use the same secondary for each antibody. A list of our anti-rabbit secondary antibodies is given through the link below, depending on the detection method and conjugation that you prefer:
https://www.abcam.com/search?AppliedFacets.FacetProductType=Secondary+antibodies&AppliedFacets.FacetReactivity=Rabbit&IsFacetsOnly=False&PageNumber=1&Keywords[0]=rabbit&X-Requested-With=XMLHttpRequest

Together these antibodies could be used to detect the presence of histone H3 in your samples as well as the methylation status of Lysine 9. To complete the spectrum, Abcam also sells an anti-histone H3 mono methyl K9 antibody (ab9045, https://www.abcam.com/histone-h3-mono-methyl-k9-antibody-chip-grade-ab9045.html). As we discussed, ab3790 (anti-KMT8/Riz1/Riz2) detects the presence of PR domain zinc finger protein 2, which is a histone methyltransferase that specifically methylates Lysine 9 of histone H3 according to SwissProt (http://www.uniprot.org/uniprot/Q13029), thus explaining its connection to the anti-histone H3 antibodies that detect the methylation status of Lysine 9

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Abcam Scientific Support

Answered on Nov 21 2012

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