Overview

  • Product name
    Anti-Histone H2A.Z antibody - ChIP Grade
    See all Histone H2A.Z primary antibodies
  • Description
    Rabbit polyclonal to Histone H2A.Z - ChIP Grade
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, ChIP, IP, CHIPseq, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Rabbit, Chicken, Cow, Human, Arabidopsis thaliana, Zebrafish
    Predicted to work with: Sheep, Xenopus laevis
  • Immunogen

    Synthetic peptide corresponding to Human Histone H2A.Z aa 100 to the C-terminus conjugated to keyhole limpet haemocyanin.
    (Peptide available as ab11681)

  • Positive control
    • ChIP: Chromatin from HeLa cells. ICC/IF: Mouse embryonic cells. HepG2 cells. WB: HeLa, NIH/3T3 and PC-12 whole cell lysate. Calf thymus histone lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab4174 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
ICC/IF 1/1000.
ChIP Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
CHIPseq Use at an assay dependent concentration. PubMed: 22196736
WB 1/1000. Detects a band of approximately 14 kDa (predicted molecular weight: 13.4 kDa).

Target

  • Function
    Variant histone H2A which replaces conventional H2A in a subset of nucleosomes. 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. May be involved in the formation of constitutive heterochromatin. May be required for chromosome segregation during cell division.
  • Sequence similarities
    Belongs to the histone H2A family.
  • Post-translational
    modifications
    Monoubiquitination of Lys-122 gives a specific tag for epigenetic transcriptional repression.
    Acetylated on Lys-5, Lys-8 and Lys-12 during interphase. Acetylation disappears at mitosis.
    Monomethylated on Lys-5 and Lys-8 by SETD6. SETD6 predominantly methylates Lys-8, lys-5 being a possible secondary site.
    Not phosphorylated.
  • Cellular localization
    Nucleus. Chromosome.
  • Information by UniProt
  • Database links
  • Alternative names
    • H2A histone family member Z antibody
    • H2A.z antibody
    • H2A/z antibody
    • H2afz antibody
    • H2AZ antibody
    • H2AZ_HUMAN antibody
    • Histone H2A.Z antibody
    • MGC117173 antibody
    see all

Images

  • Changes in nucleosome occupancy upon LPS induction at a putative distal enhancer and promoter of IL1A, kinetics of nucleosome removal, and changes in histone modifications.

    ChIP experiments with antibodies against H3 (dark blue bars), H2A.Z (light blue), H3K4me1 (green), H3K4me3 (yellow) and H3K27ac (red). For these experiments cross-linked chromatin was lightly digested with MNase before incubation with the respective antibodies to increase resolution of the ChIP signal and the data was normalized to a region in the ORF of RPL4. Changes upon LPS induction in histone binding and histone modifications at the enhancers and promoters of IL12B and IL1A as well as at a control region in the GAPDH pseudo gene are shown as fold over levels found before induction. For H3K27ac the changes 1.5 h after LPS induction, and for all other histone variants and modifications the changes after 3 h of induction are shown. The error bars show the SEM of at least 3 independent experiments. Statistical significance of the changes in H3K4me3 and H3K27ac upon LPS induction compared to levels found prior to induction determined by Student's T-tests is indicated (*P<0.05; **P<0.01).

  • Chromatin was prepared from HeLa (Human epithelial cell line from cervix adenocarcinoma) 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  ab4174 (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.

  • Lanes 1-2 : Anti-Histone H2A.Z antibody - ChIP Grade (ab4174) at 1/500 dilution
    Lanes 3-4 : Anti-Histone H2A.Z antibody - ChIP Grade (ab4174) at 1/1000 dilution

    Lanes 1 & 3 : Calf thymus histone lysate
    Lanes 2 & 4 : Calf thymus histone lysate with Human Histone H2A.Z peptide (ab11681) at 1 µg/ml

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

    Performed under reducing conditions.

    Predicted band size: 13.4 kDa

  • Staining of Histone H2A.Z in mouse embryonic cells. The fixation is 2% PFA, and permeabilization is PBS 0.5% triton BSA. The dilution used was 1 in 100 (but it could be used at 1/200 to 1/300).

    Red  = H2A.Z
    Blue  = toto3 for the DNA

  • All lanes : Anti-Histone H2A.Z antibody - ChIP Grade (ab4174) at 1 µg/ml

    Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg
    Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg
    Lane 3 : NIH/3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg
    Lane 4 : PC-12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 13.4 kDa


    Exposure time: 3 minutes
  • This image was kindly supplied as part of the review submitted by Dr Geza Fejes-Toth.
  • ICC/IF image of ab4174 stained HepG2 (Human liver hepatocellular carcinoma cell line) cells.

    The cells were fixed with 100% methanol (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab4174, 5 µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit 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.

  • All lanes : Anti-Histone H2A.Z antibody - ChIP Grade (ab4174) at 1/1000 dilution

    Lane 1 : Native recombinant octamers K562 (Human chronic myelogenous leukemia cell line from bone marrow) cells at 3 µg
    Lane 2 : Native recombinant octamers K562 cells at 1.5 µg
    Lane 3 : Recombinant Human octamers containing H2A at 1 µg
    Lane 4 : Recombinant Human octamers containing H2A at 0.5 µg
    Lane 5 : Recombinant Human octamers containing H2A.Z.2.1 at 0.5 µg
    Lane 6 : Recombinant Human octamers containing H2A.Z.1 at 0.5 µg

    Secondary
    All lanes : HRP-conjugated donkey anti-rabbit IgG polyclonal at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 13.4 kDa


    Exposure time: 5 minutes


    Blocked with 3% BSA for 1 hour at 20°C.

    Primary incubation in TBS tween + 3% BSA at 20°C for 1 hour.

    See Abreview

References

This product has been referenced in:
  • Suen DF  et al. The Deubiquitinase OTU5 Regulates Root Responses to Phosphate Starvation. Plant Physiol N/A:N/A (2018). Read more (PubMed: 29301952) »
  • He Y  et al. Androgen receptor splice variants bind to constitutively open chromatin and promote abiraterone-resistant growth of prostate cancer. Nucleic Acids Res 46:1895-1911 (2018). Read more (PubMed: 29309643) »
See all 95 Publications for this product

Customer reviews and Q&As

1-10 of 39 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - nuclear (Mouse embryonic fibroblasts)
Loading amount
25 µg
Specification
Mouse embryonic fibroblasts
Gel Running Conditions
Reduced Denaturing (4-20% Tris-Gly gel)
Blocking step
LI-COR® Odyssey® Blocking Buffer as blocking agent for 45 minute(s) · Concentration: 50% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Dec 12 2011

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Saccharomyces cerevisiae Cell lysate - whole cell (WT and htz1 delta (H2A.Z deletion ) cells)
Gel Running Conditions
Non-reduced Denaturing (15% gel)
Loading amount
1e+007 cells
Specification
WT and htz1 delta (H2A.Z deletion ) cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 30°C

Daisuke Takahashi

Verified customer

Submitted Aug 13 2018

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Permeabilization
Yes - 0.5% Triton 5 minutes
Specification
HeLa
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jan 25 2018

Application
Immunohistochemistry (Resin sections)
Sample
Astatotilapia burtoni Tissue sections (Brain preopticarea embedded in LRWhite)
Specification
Brain preopticarea embedded in LRWhite

Sebastian Alvarado

Verified customer

Submitted May 03 2016

Application
Western blot
Loading amount
0.5 µg
Gel Running Conditions
Reduced Denaturing (4-20% TGX (BioRad))
Sample
Human Purified protein (Recombinant octamers & purified K562 cell histones)
Specification
Recombinant octamers & purified K562 cell histones
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 20°C

Dr. Ragnhild Eskeland

Verified customer

Submitted Jan 27 2015

Application
ChIP
Detection step
Other
Sample
Mouse Cell lysate - whole cell (bone marrow derived mouse macrophages)
Specification
bone marrow derived mouse macrophages
Negative control
IgG
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: formaldehyde 1% final
Positive control
Pu.1 antibody

Dr. Silvia Bonifacio

Verified customer

Submitted Oct 24 2014

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing (15%)
Sample
Arabidopsis thaliana Tissue lysate - nuclear (7dps seedlings)
Specification
7dps seedlings
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Sep 25 2014

Application
ChIP
Detection step
Real-time PCR
Sample
Mouse Cell lysate - whole cell (Embryonic Stem Cells)
Specification
Embryonic Stem Cells
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: formaldehyde

Mr. Dan Stummer

Verified customer

Submitted Mar 29 2014

Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Reduced Denaturing
Sample
Mouse Cell lysate - whole cell (Murine embryonic stem cells)
Specification
Murine embryonic stem cells
Treatment
vehicle control (EtOH) or all-trans retinoic acid (RA)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Mr. Dan Stummer

Verified customer

Submitted Mar 28 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Sample
Human Cell lysate - nuclear (haematopoietic)
Specification
haematopoietic
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: formaldehyde
Detection step
Real-time PCR
Positive control
PU.1
Negative control
IVL

Abcam user community

Verified customer

Submitted Dec 26 2012

1-10 of 39 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Sign up