Anti-HMGB1抗体- ChIP Grade (ab18256)

概述

  • 产���名称Anti-HMGB1抗体- ChIP Grade
    参阅全部 HMGB1 一抗
  • 描述
    兔多克隆抗体to HMGB1 - ChIP Grade
  • 经测试应用适用于: IHC-FoFr, ICC/IF, IHC-P, ChIP, IHC-Fr, ICC, ELISA, IP, WBmore details
  • 种属反应性
    与反应: Mouse, Rat, Human
    预测可用于: Rabbit, Cow
  • 免疫原

    Synthetic peptide conjugated to KLH derived from within residues 150 to the C-terminus of Human HMGB1.

    (Peptide available as ab18650.)

  • 阳性对照
    • This antibody gave a positive signal in the following whole cell lysates: HeLa (Human epithelial carcinoma cell line) Jurkat (Human T cell lymphoblast-like cell line) A431 (Human epithelial carcinoma cell line) HEK 293 (Human embryonic kidney cell line) NIH 3T3 (Mouse embryonic fibroblast cell line) MEF1 (Mouse embryonic fibroblast cell line) PC12 (Rat adrenal pheochromocytoma cell line) This antibody gave a positive signal in the following nuclear lysate: HeLa nuclear lysate
  • 常规说明

     

     

     

性能

应用

Our Abpromise guarantee covers the use of ab18256 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
IHC-FoFr Use at an assay dependent concentration.
ICC/IF Use a concentration of 1 µg/ml.
IHC-P 1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
ChIP Use at an assay dependent concentration.
IHC-Fr 1/100.
ICC Use at an assay dependent concentration.
ELISA Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 29 kDa (predicted molecular weight: 25 kDa).

靶标

  • 功能Multifunctional redox sensitive protein with various roles in different cellular compartments. In the nucleus is one of the major chromatin-associated non-histone proteins and acts as a DNA chaperone involved in replication, transcription, chromatin remodeling, V(D)J recombination, DNA repair and genome stability. Proposed to be an universal biosensor for nucleic acids. Promotes host inflammatory response to sterile and infectious signals and is involved in the coordination and integration of innate and adaptive immune responses. In the cytoplasm functions as sensor and/or chaperone for immunogenic nucleic acids implicating the activation of TLR9-mediated immune responses, and mediates autophagy. Acts as danger associated molecular pattern (DAMP) molecule that amplifies immune responses during tissue injury. Released to the extracellular environment can bind DNA, nucleosomes, IL-1 beta, CXCL12, AGER isoform 2/sRAGE, lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and activates cells through engagement of multiple surface receptors. In the extracellular compartment fully reduced HMGB1 (released by necrosis) acts as a chemokine, disulfide HMGB1 (actively secreted) as a cytokine, and sulfonyl HMGB1 (released from apoptotic cells) promotes immunological tolerance (PubMed:23519706, PubMed:23446148, PubMed:23994764, PubMed:25048472). Has proangiogdenic activity (By similarity). May be involved in platelet activation (By similarity). Binds to phosphatidylserine and phosphatidylethanolamide (By similarity). Bound to RAGE mediates signaling for neuronal outgrowth (By similarity). May play a role in accumulation of expanded polyglutamine (polyQ) proteins such as huntingtin (HTT) or TBP (PubMed:23303669, PubMed:25549101).
    Nuclear functions are attributed to fully reduced HGMB1. Associates with chromatin and binds DNA with a preference to non-canonical DNA structures such as single-stranded DNA, DNA-containing cruciforms or bent structures, supercoiled DNA and ZDNA. Can bent DNA and enhance DNA flexibility by looping thus providing a mechanism to promote activities on various gene promoters by enhancing transcription factor binding and/or bringing distant regulatory sequences into close proximity (PubMed:20123072). May have an enhancing role in nucleotide excision repair (NER) (By similarity). However, effects in NER using in vitro systems have been reported conflictingly (PubMed:19446504, PubMed:19360789). May be involved in mismatch repair (MMR) and base excision repair (BER) pathways (PubMed:15014079, PubMed:16143102, PubMed:17803946). May be involved in double strand break repair such as non-homologous end joining (NHEJ) (By similarity). Involved in V(D)J recombination by acting as a cofactor of the RAG complex: acts by stimulating cleavage and RAG protein binding at the 23 bp spacer of conserved recombination signal sequences (RSS) (By similarity). In vitro can displace histone H1 from highly bent DNA (By similarity). Can restructure the canonical nucleosome leading to relaxation of structural constraints for transcription factor-binding (By similarity). Enhances binding of sterol regulatory element-binding proteins (SREBPs) such as SREBF1 to their cognate DNA sequences and increases their transcriptional activities (By similarity). Facilitates binding of TP53 to DNA (PubMed:23063560). Proposed to be involved in mitochondrial quality control and autophagy in a transcription-dependent fashion implicating HSPB1; however, this function has been questioned (By similarity). Can modulate the activity of the telomerase complex and may be involved in telomere maintenance.
    In the cytoplasm proposed to dissociate the BECN1:BCL2 complex via competitive interaction with BECN1 leading to autophagy activation (PubMed:20819940). Involved in oxidative stress-mediated autophagy (PubMed:21395369). Can protect BECN1 and ATG5 from calpain-mediated cleavage and thus proposed to control their proautophagic and proapoptotic functions and to regulate the extent and severity of inflammation-associated cellular injury (By similarity). In myeloid cells has a protective role against endotoxemia and bacterial infection by promoting autophagy (By similarity). Involved in endosomal translocation and activation of TLR9 in response to CpG-DNA in macrophages.
    In the extracellular compartment (following either active secretion or passive release) involved in regulation of the inflammatory response. Fully reduced HGMB1 (which subsequently gets oxidized after release) in association with CXCL12 mediates the recruitment of inflammatory cells during the initial phase of tissue injury; the CXCL12:HMGB1 complex triggers CXCR4 homodimerization (PubMed:22370717). Induces the migration of monocyte-derived immature dendritic cells and seems to regulate adhesive and migratory functions of neutrophils implicating AGER/RAGE and ITGAM (By similarity). Can bind to various types of DNA and RNA including microbial unmethylated CpG-DNA to enhance the innate immune response to nucleic acids. Proposed to act in promiscuous DNA/RNA sensing which cooperates with subsequent discriminative sensing by specific pattern recognition receptors (By similarity). Promotes extracellular DNA-induced AIM2 inflammasome activation implicating AGER/RAGE (PubMed:24971542). Disulfide HMGB1 binds to transmembrane receptors, such as AGER/RAGE, TLR2, TLR4 and probably TREM1, thus activating their signal transduction pathways. Mediates the release of cytokines/chemokines such as TNF, IL-1, IL-6, IL-8, CCL2, CCL3, CCL4 and CXCL10 (PubMed:12765338, PubMed:18354232, PubMed:19264983, PubMed:20547845, PubMed:24474694). Promotes secretion of interferon-gamma by macrophage-stimulated natural killer (NK) cells in concert with other cytokines like IL-2 or IL-12 (PubMed:15607795). TLR4 is proposed to be the primary receptor promoting macrophage activation and signaling through TLR4 seems to implicate LY96/MD-2 (PubMed:20547845). In bacterial LPS- or LTA-mediated inflammatory responses binds to the endotoxins and transfers them to CD14 for signaling to the respective TLR4:LY96 and TLR2 complexes (PubMed:18354232, PubMed:21660935, PubMed:25660311). Contributes to tumor proliferation by association with ACER/RAGE (By similarity). Can bind to IL1-beta and signals through the IL1R1:IL1RAP receptor complex (PubMed:18250463). Binding to class A CpG activates cytokine production in plasmacytoid dendritic cells implicating TLR9, MYD88 and AGER/RAGE and can activate autoreactive B cells. Via HMGB1-containing chromatin immune complexes may also promote B cell responses to endogenous TLR9 ligands through a B-cell receptor (BCR)-dependent and ACER/RAGE-independent mechanism (By similarity). Inhibits phagocytosis of apoptotic cells by macrophages; the function is dependent on poly-ADP-ribosylation and involves binding to phosphatidylserine on the cell surface of apoptotic cells (By similarity). In adaptive immunity may be involved in enhancing immunity through activation of effector T cells and suppression of regulatory T (TReg) cells (PubMed:15944249, PubMed:22473704). In contrast, without implicating effector or regulatory T-cells, required for tumor infiltration and activation of T-cells expressing the lymphotoxin LTA:LTB heterotrimer thus promoting tumor malignant progression (By similarity). Also reported to limit proliferation of T-cells (By similarity). Released HMGB1:nucleosome complexes formed during apoptosis can signal through TLR2 to induce cytokine production (PubMed:19064698). Involved in induction of immunological tolerance by apoptotic cells; its pro-inflammatory activities when released by apoptotic cells are neutralized by reactive oxygen species (ROS)-dependent oxidation specifically on Cys-106 (PubMed:18631454). During macrophage activation by activated lymphocyte-derived self apoptotic DNA (ALD-DNA) promotes recruitment of ALD-DNA to endosomes.
  • 组织特异性Ubiquituous. Expressed in platelets (PubMed:11154118).
  • 序列相似性Belongs to the HMGB family.
    Contains 2 HMG box DNA-binding domains.
  • 结构域HMG box 2 mediates proinflammatory cytokine-stimulating activity and binding to TLR4 (PubMed:12765338, PubMed:20547845). However, not involved in mediating immunogenic activity in the context of apoptosis-induced immune tolerance (PubMed:24474694).
    The acidic C-terminal domain forms a flexible structure which can reversibly interact intramolecularily with the HMG boxes and modulate binding to DNA and other proteins (PubMed:23063560).
  • 翻译后修饰Phosphorylated at serine residues. Phosphorylation in both NLS regions is required for cytoplasmic translocation followed by secretion (PubMed:17114460).
    Acetylated on multiple sites upon stimulation with LPS (PubMed:22801494). Acetylation on lysine residues in the nuclear localization signals (NLS 1 and NLS 2) leads to cytoplasmic localization and subsequent secretion (By similarity). Acetylation on Lys-3 results in preferential binding to DNA ends and impairs DNA bending activity.
    Reduction/oxidation of cysteine residues Cys-23, Cys-45 and Cys-106 and a possible intramolecular disulfide bond involving Cys-23 and Cys-45 give rise to different redox forms with specific functional activities in various cellular compartments: 1- fully reduced HMGB1 (HMGB1C23hC45hC106h), 2- disulfide HMGB1 (HMGB1C23-C45C106h) and 3- sulfonyl HMGB1 (HMGB1C23soC45soC106so).
    Poly-ADP-ribosylated by PARP1 when secreted following stimulation with LPS.
    In vitro cleavage by CASP1 is liberating a HMG box 1-containing peptide which may mediate immunogenic activity; the peptide antagonizes apoptosis-induced immune tolerance (PubMed:24474694). Can be proteolytically cleaved by a thrombin:thrombomodulin complex; reduces binding to heparin and proinflammatory activities.
  • 细胞定位Nucleus. Chromosome. Cytoplasm. Secreted. Cell membrane. Endosome. Endoplasmic reticulum-Golgi intermediate compartment. In basal state predominantly nuclear. Shuttles between the cytoplasm and the nucleus (PubMed:12231511, PubMed:17114460). Translocates from the nucleus to the cytoplasm upon autophagy stimulation (PubMed:20819940). Release from macrophages in the extracellular milieu requires the activation of NLRC4 or NLRP3 inflammasomes (By similarity). Passively released to the extracellular milieu from necrotic cells by diffusion, involving the fully reduced HGMB1 which subsequently gets oxidized (PubMed:19811284). Also released from apoptic cells (PubMed:16855214, PubMed:18631454). Active secretion from a variety of immune and non-immune cells such as macrophages, monocytes, neutrophils, dendritic cells and natural killer cells in response to various stimuli such as LPS and cytokines involves a nonconventional secretory process via secretory lysosomes (PubMed:12231511, PubMed:14532127, PubMed:15944249). Secreted by plasma cells in response to LPS (By similarity). Found on the surface of activated platelets (PubMed:11154118).
  • Information by UniProt
  • 数据库链接
  • 别名
    • Amphoterin antibody
    • Chromosomal protein, nonhistone, HMG1 antibody
    • DKFZp686A04236 antibody
    • High mobility group 1 antibody
    • High mobility group box 1 antibody
    • High mobility group protein 1 antibody
    • High mobility group protein B1 antibody
    • high-mobility group (nonhistone chromosomal) protein 1 antibody
    • HMG-1 antibody
    • HMG1 antibody
    • HMG3 antibody
    • HMGB 1 antibody
    • HMGB1 antibody
    • HMGB1_HUMAN antibody
    • NONHISTONE CHROMOSOMAL PROTEIN HMG1 antibody
    • SBP 1 antibody
    • Sulfoglucuronyl carbohydrate binding protein antibody
    see all

Anti-HMGB1 antibody - ChIP Grade 图像

  • Image courtesy of Human Protein Atlas

    Paraffin embedded sections of human liver were incubated with ab18256 (1/1000 dilution) for 30 minutes at room temperature. Heat induced antigen retrieval was performed in citrate buffer pH 6.

    ab18256 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 images can be found www.proteinatlas.org

  • All lanes : Anti-HMGB1 antibody - ChIP Grade (ab18256) at 1 µg/ml

    Lane 1 : NIH 3T3 whole cell lysate (ab7179)
    Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
    Lane 3 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

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

    Performed under reducing conditions.

    Predicted band size : 25 kDa
    Observed band size : 29 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 59 kDa. We are unsure as to the identity of these extra bands.
  • ICC/IF image of ab18256 stained Hela cells. The cells were 4% PFA fixed (10 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 (ab18256, 5µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - HandL, 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-HMGB1 antibody - ChIP Grade (ab18256) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : Jurkat whole cell lysate (ab7899)
    Lane 3 : A431 whole cell lysate (ab7909)
    Lane 4 : HEK293 whole cell lysate (ab7902)

    Lysates/proteins at 10 µg per lane.

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

    Performed under reducing conditions.

    Predicted band size : 25 kDa
    Observed band size : 29 kDa (why is the actual band size different from the predicted?)
  • ab18256 staining HMGB1 in mouse liver tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 15% serum for 60 minutes at 20°C; antigen retrieval was by heat mediation. Samples were incubated with primary antibody (1/1000 in TBS) for 18 hours at 20°C. An Alexa Fluor® 647-conjugated goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

    See Abreview

  • ICC/IF image of ab18256 stained HeLa cells. The cells were 4% PFA fixed (10 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 (ab18256, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 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. This antibody also gave a positive result in 4% PFA fixed (10 min) Hek293, HepG2 and MCF7 cells at 1µg/ml.
  • ab18256 staining HMGB1 in Human stomach tissue sections by IHC-Fr (Immunohistochemistry - Frozen sections). Tissue samples were fixed with acetone and blocked with 5% serum for 1 hour at 25°C. Samples were incubated with primary antibody 1/500 in blocking buffer for 1 hour at 25°C. An undiluted HRP-conjugated Goat polyclonal to rabbit IgG was used as secondary antibody.

    See Abreview

  • All lanes : Anti-HMGB1 antibody - ChIP Grade (ab18256) at 1/1000 dilution

    Lane 1 : Rat brain whole tissue lysate - infused with asf for 1 week
    Lane 2 : Rat brain whole tissue lysate - infused with LPS for 1 week
    Lane 3 : Rat brain whole tissue lysate - infused with acsf for 8 weeks
    Lane 4 : Rat brain whole tissue lysate - infused with LPS for 8 weeks
    Lane 5 : Rat brain whole tissue lysate - infused with LPS for 4 weeks
    Lane 6 : Rat brain whole tissue lysate -infused with LPS for 4 weeks, after 2 weeks of LPS infusion were treated with neramexane for the next 2 weeks
    Lane 7 : Rat brain whole tissue lysate - infused with LPS for 4 weeks, after 2 weeks of LPS infusion were treated with memantine for the next 2 weeks.

    Lysates/proteins at 40 µg per lane.

    Secondary
    Biotinylated Goat anti-rabbit IgG
    Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 25 kDa


    Exposure time : 30 seconds

    This image is courtesy of an Abreview submitted by Dr Francesca Cerbai

    See Abreview

  • ab18256 used in Direct ELISA in NIH 3T3 murine fibroblasts. Primary antibody used at a 1/1000 dilution for 16 hours at 4°C. The secondary antibody is an AP-conjugated Goat anti-rabbit used at a 1/1000 dilution. A blocking step was performed using 5% BSA for 1 hour.

    See Abreview


  • Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 25 kDa


    Exposure time : 2 minutes
  • ab18256 staining HMGB1 in murine kidney tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).
    Tissue was fixed in formaldehyde and a heat mediated antigen retrieval step was performed using citrate EDTA buffer pH 6.2. Samples were then blocked, then incubated with ab18256 at a 1/1000 dilution for 1 hour. The secondary used was a goat anti-rabbit IgG conjugated to HRP at a 1/1000 dilution.

    See Abreview

Anti-HMGB1 antibody - ChIP Grade (ab18256)参考文献

This product has been referenced in:
  • Xiong X  et al. Glycyrrhizin protects against focal cerebral ischemia via inhibition of T cell activity and HMGB1-mediated mechanisms. J Neuroinflammation 13:241 (2016). WB ; Rat . Read more (PubMed: 27609334) »
  • Norouzitallab P  et al. Non-lethal heat shock induces HSP70 and HMGB1 protein production sequentially to protect Artemia franciscana against Vibrio campbellii. Fish Shellfish Immunol 42:395-9 (2015). Read more (PubMed: 25463291) »

See all 76 Publications for this product

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Application Immunocytochemistry/ Immunofluorescence
Sample Mouse Cell (Pulmonary microvascular)
Permeabilization Yes - 0.5% Triton in PBS
Specification Pulmonary microvascular
Blocking step BSA/NGS as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2.5% · Temperature: 25°C
Fixative Methanol:Acetone
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提交于 Nov 15 2016

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Human Tissue sections (Liver)
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: citrate pH6.0
Permeabilization No
Specification Liver
Blocking step Leica Novolink kit (protein and peroxidase blocks) as blocking agent for 10 minute(s) · Concentration: 0µg/mL
Fixative Formaldehyde
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提交于 Aug 17 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Mouse Tissue lysate - whole (Whole lung tissue)
Gel Running Conditions Reduced Denaturing (4-12% BT Gel)
Loading amount 25 µg
Specification Whole lung tissue
Blocking step Odyssey Blocking Buffer (PBS) as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: RT°C
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Jose Vazquez-Medina

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提交于 Aug 17 2016

Abcam has not validated the combination of species/application used in this Abreview.
Application Blocking
Sample Mouse Cell (MLO-Y4)
Specification MLO-Y4
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提交于 Mar 21 2016

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Mouse Tissue sections (liver)
Antigen retrieval step Heat mediated
Permeabilization No
Specification liver
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 15% · Temperature: 20°C
Fixative Formaldehyde
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提交于 Dec 18 2015

That could be a fragment, post-caspase cleavage, though it does not look like the band intensity correlates with treatment. The UniProt entry for human HMGB1, http://www.uniprot.org/uniprot/P09429, includes an annotation in the Function/sites section r...

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Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (skeletal muscle)
Permeabilization Yes - 0.1% Triton X100
Specification skeletal muscle
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 22°C
Fixative Paraformaldehyde
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提交于 Aug 05 2015

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Rhesus monkey Tissue sections (ovary)
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Reveal Decloacker (Biocare Medical)
Permeabilization No
Specification ovary
Blocking step Avidin/Biotin blocking kit (Vector) then TBS + 10% goat serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 22°C
Fixative Modified Davidson's Fixative (EMS)
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Mrs. Lindsay Reustle

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提交于 May 27 2015

Application Western blot
Loading amount 100 µg
Gel Running Conditions Reduced Denaturing (10% Tris-Glycine)
Sample Cow Cell lysate - whole cell (MDBK cells)
Specification MDBK cells
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 22°C
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提交于 Mar 09 2015

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