Anti-Histone H2A抗体- ChIP Grade (ab18255)

概述

  • 产品名称Anti-Histone H2A抗体- ChIP Grade
    参阅全部 Histone H2A 一抗
  • 描述
    兔多克隆抗体to Histone H2A - ChIP Grade
  • 经测试应用适用于: ICC/IF, IHC-P, WB, IP, ChIPmore details
  • 种属反应性
    与反应: Mouse, Human
  • 免疫原

    Synthetic peptide conjugated to KLH derived from within residues 100 to the C-terminus of Human Histone H2A.

  • 阳性对照
    • Hela nuclear extract, Calf Thymus Histone Preparation

性能

应用

Our Abpromise guarantee covers the use of ab18255 in the following tested applications.

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

应用 Ab评论 说明
ICC/IF 1/200. see Abreview submitted by Kirk McManus
IHC-P 1/150. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 14 kDa (predicted molecular weight: 14 kDa).
IP Use a concentration of 5 µg/ml.
ChIP Use at an assay dependent concentration. Every new batch of this antibody is tested at Abcam in ChIP

靶标

  • 功能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.
  • 序列相似性Belongs to the histone H2A family.
  • 翻译后修饰The chromatin-associated form is phosphorylated on Thr-121 during mitosis.
    Deiminated on Arg-4 in granulocytes upon calcium entry.
    Monoubiquitination of Lys-120 by RING1 and RNF2/RING2 complex gives a specific tag for epigenetic transcriptional repression and participates in X chromosome inactivation of female mammals. It is involved in the initiation of both imprinted and random X inactivation. Ubiquitinated H2A is enriched in inactive X chromosome chromatin. Ubiquitination of H2A functions downstream of methylation of 'Lys-27' of histone H3. Monoubiquitination of Lys-120 by RNF2/RING2 can also be induced by ultraviolet and may be involved in DNA repair. Following DNA double-strand breaks (DSBs), it is ubiquitinated through 'Lys-63' linkage of ubiquitin moieties by the E2 ligase UBE2N and the E3 ligases RNF8 and RNF168, leading to the recruitment of repair proteins to sites of DNA damage. Monoubiquitination and ionizing radiation-induced 'Lys-63'-linked ubiquitination are distinct events.
    Phosphorylation on Ser-2 is enhanced during mitosis. Phosphorylation on Ser-2 by RPS6KA5/MSK1 directly represses transcription. Acetylation of H3 inhibits Ser-2 phosphorylation by RPS6KA5/MSK1.
    Symmetric dimethylation on Arg-4 by the PRDM1/PRMT5 complex may play a crucial role in the germ-cell lineage.
  • 细胞定位Nucleus. Chromosome.
  • Information by UniProt
  • 数据库链接
  • 别名
    • H2a 615 antibody
    • H2A antibody
    • H2A GL101 antibody
    • H2A histone family member A antibody
    • H2A.1 antibody
    • H2A.2 antibody
    • H2A/a antibody
    • H2A/m antibody
    • H2A/O antibody
    • H2A/q antibody
    • H2A1B_HUMAN antibody
    • H2AFA antibody
    • H2AFE antibody
    • H2AFL antibody
    • H2AFM antibody
    • H2AFO antibody
    • H2AFQ antibody
    • HIST1H2AE antibody
    • HIST1H2AJ antibody
    • HIST2H2AA antibody
    • HIST2H2AA3 antibody
    • HIST2H2AB antibody
    • HIST2H2AC antibody
    • Histone 1 H2ae antibody
    • Histone 2 H2aa3 antibody
    • Histone 2 H2ab antibody
    • Histone 2 H2ac antibody
    • Histone H2A type 1 B antibody
    • Histone H2A type 1 C antibody
    • Histone H2A type 1 E antibody
    • Histone H2A type 1 J antibody
    • Histone H2A type 1-B/E antibody
    • Histone H2A.2 antibody
    • Histone H2A/a antibody
    • Histone H2A/m antibody
    • MGC74460 antibody
    see all

Anti-Histone H2A antibody - ChIP Grade 图像

  • All lanes : Anti-Histone H2A antibody - ChIP Grade (ab18255) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg
    Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg
    Lane 3 : Histone H2A Recombinant Protein at 0.1 µg
    Lane 4 : Histone H3.1 Recombinant Protein at 0.1 µg
    Lane 5 : Histone H4 Recombinant Protein at 0.1 µg

    Secondary
    Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 14 kDa
    Observed band size : 17 kDa (why is the actual band size different from the predicted?)


    Exposure time : 3 minutes
  • Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 6µl of ab18255 (blue), and 20µl 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.

  • Image courtesy of Human Protein Atlas

    ab18255 staining histone H2A in human testis, showing a distinct and strong nuclear staining pattern at cells in ductus seminiferus. Paraffin embedded human skin tissue was incubated with ab18255 (1/1200 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.

    ab18255 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org

    .
  • Histone H2A - ChIP Grade was immunoprecipitated using 0.5mg HeLa whole cell extract, 5µg of Rabbit polyclonal to 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 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab18255.

    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

    Band: 14kDa, non specific bands - 42kDa: We are unsure as to the identity of this extra band; Histone H2A - ChIP Grade

  • All lanes : Anti-Histone H2A antibody - ChIP Grade (ab18255) at 1 µg/ml

    Lane 1 : HeLa lysate at 20 µg
    Lane 2 : HeLa nuclear lysate at 20 µg
    Lane 3 : Calf thymus histone lysate at 20 µg
    Lane 4 : HeLa lysate at 1 µg/ml with Human Histone H2A peptide (ab19751) at 1 µg/ml
    Lane 5 : HeLa nuclear lysate at 1 µg/ml with Human Histone H2A peptide (ab19751) at 1 µg/ml
    Lane 6 : Calf thymus histone lysate at 1 µg/ml with Human Histone H2A peptide (ab19751) at 1 µg/ml


    Predicted band size : 14 kDa
    Observed band size : 14 kDa
    Additional bands at : 22 kDa (possible cross reactivity).

    ab18255 is partially blocked by the immunizing peptide ab19751. There is an additional band at 22kDa in HeLa lysate which is attributed to cross-reactivity.

  • ICC/IF image of ab18255 stained HeLa cells. The cells were 100% methanol fixed (5 min) then permeabilised using 0.1% PBS-Triton and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to further permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab18255 at 1µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

  • All lanes : Anti-Histone H2A antibody - ChIP Grade (ab18255) at 1/1000 dilution

    Lane 1 : Native recombinant octamers K562 cells
    Lane 2 : Recombinant Human octamers containing H2A
    Lane 3 : Recombinant Human octamers containing H2A.Z.2.1
    Lane 4 : Recombinant Human octamers containing H2A.Z.1

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    HRP-conjugated donkey anti-rabbit IgG polyclonal at 1/10000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 14 kDa
    Observed band size : 15 kDa (why is the actual band size different from the predicted?)


    Exposure time : 5 minutes

    This image is courtesy of an Abreview submitted by Ragnhild Eskeland

    See Abreview

Anti-Histone H2A antibody - ChIP Grade (ab18255)参考文献

This product has been referenced in:
  • Kocylowski MK  et al. Ubiquitin-H2AX fusions render 53BP1 recruitment to DNA damage sites independent of RNF8 or RNF168. Cell Cycle 14:1748-58 (2015). WB ; Human . Read more (PubMed: 25695757) »
  • Gao W  et al. Plasma membrane proteomic analysis of human Gastric Cancer tissues: revealing flotillin 1 as a marker for Gastric Cancer. BMC Cancer 15:367 (2015). WB ; Human . Read more (PubMed: 25948494) »

See all 29 Publications for this product

Product Wall

Application Western blot
Sample Human Cell lysate - whole cell (U2OS)
Gel Running Conditions Reduced Denaturing (4-20%)
Loading amount 5 µg
Specification U2OS
Username

Abcam user community

Verified customer

提交于 Oct 05 2016

Application Western blot
Loading amount 0.5 µg
Gel Running Conditions Reduced Denaturing (Criterion TGX 4-20% (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
Username

Dr. Ragnhild Eskeland

Verified customer

提交于 Jan 27 2015

Application Western blot
Loading amount 10000 cells
Gel Running Conditions Reduced Denaturing (4-12% gradient)
Sample Mouse Cell lysate - whole cell (MEFs)
Specification MEFs
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

提交于 Dec 04 2014

Application Western blot
Loading amount 10000 cells
Gel Running Conditions Reduced Denaturing (4-12% gradient)
Sample Human Cell lysate - nuclear (Huh7 liver)
Specification Huh7 liver
Treatment Infected with dengue virus 24 hours
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

提交于 Nov 18 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (Ntera2 cells)
Loading amount 20 µg
Specification Ntera2 cells
Gel Running Conditions Reduced Denaturing (15% gel)
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

提交于 May 29 2012

ab11175 and ab18255 were never used togteher in Sandwich ELISA. These however can be used in Indirect ELISA.

The sELISA application totally based on difference in epiotopes, the antibodies recognize means the epitopes should not interfere, ab...

Read More
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 30 µg
Specification Mouse embryonic fibroblasts
Gel Running Conditions Reduced Denaturing (4-16% Tris-Gly gel)
Blocking step LI-COR® Odyssey® Blocking Buffer as blocking agent for 45 minute(s) · Concentration: 50% · Temperature: RT°C
Username

Abcam user community

Verified customer

提交于 Feb 23 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Mouse Cell lysate - whole cell (Mouse ES cells)
Loading amount 50 µg
Specification Mouse ES cells
Gel Running Conditions Reduced Denaturing (15% SDS-PAGE)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C
Username

Abcam user community

Verified customer

提交于 Nov 29 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (Hela Cells)
Loading amount 50 µg
Specification Hela Cells
Gel Running Conditions Reduced Denaturing (15% SDS-PAGE)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C
Username

Abcam user community

Verified customer

提交于 Nov 29 2011

1-10 of 18 Abreviews or Q&A

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