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Epigenetics and Nuclear Signaling Histones HMGs
使用敲除细胞株进行验证重组RabMAb

重组Anti-HMGB1抗体[EPR3507] (ab79823)

  • Datasheet
  • SDS
Reviews (7)Q&A (1)References (195)

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Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823)
  • Immunocytochemistry/ Immunofluorescence - Anti-HMGB1 antibody [EPR3507] (ab79823)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGB1 antibody [EPR3507] (ab79823)
  • Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823)
  • Immunocytochemistry/ Immunofluorescence - Anti-HMGB1 antibody [EPR3507] (ab79823)
  • Immunocytochemistry/ Immunofluorescence - Anti-HMGB1 antibody [EPR3507] (ab79823)
  • Immunocytochemistry/ Immunofluorescence - Anti-HMGB1 antibody [EPR3507] (ab79823)
  • Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823)
  • Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823)
  • Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGB1 antibody [EPR3507] (ab79823)
  • Flow Cytometry (Intracellular) - Anti-HMGB1 antibody [EPR3507] (ab79823)
  • Anti-HMGB1 antibody [EPR3507] (ab79823)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR3507] to HMGB1
  • Suitable for: Flow Cyt (Intra), ICC/IF, WB, IHC-P
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

Alexa Fluor® 405 Alexa Fluor® 488 Alexa Fluor® 555 Alexa Fluor® 594 Alexa Fluor® 647 APC Carrier Free HRP PE

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概述

  • 产品名称

    Anti-HMGB1抗体[EPR3507]
    参阅全部 HMGB1 一抗
  • 描述

    兔单克隆抗体[EPR3507] to HMGB1
  • 宿主

    Rabbit
  • 经测试应用

    适用于: Flow Cyt (Intra), ICC/IF, WB, IHC-Pmore details
    不适用于: IP
  • 种属反应性

    与反应: Mouse, Rat, Human
  • 免疫原

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • 阳性对照

    • WB: SK-BR-3, HeLa, HepG2, Jurkat and Wild-type HAP1 whole cell lysates; rat brain tissue lysate. IHC-P: Human kidney and tonsil tissues. ICC/IF: DU 145, HeLa, HAP1 wildtype and RAW 264.7 cells. Flow Cyt (intra): HeLa cells.
  • 常规说明

    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.

性能

  • 形式

    Liquid
  • 存放说明

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Stable for 12 months at -20°C.
  • 存储溶液

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

    Protein A purified
  • 克隆

    单克隆
  • 克隆编号

    EPR3507
  • 同种型

    IgG
  • 研究领域

    • Epigenetics and Nuclear Signaling
    • Histones
    • HMGs
    • Epigenetics and Nuclear Signaling
    • Transcription
    • Domain Families
    • HMG Box
    • Microbiology
    • Organism
    • Virus
    • RNA Virus
    • ssRNA positive strand virus
    • SARS Coronavirus
    • Neuroscience
    • Processes

相关产品

  • Alternative Versions

    • Alexa Fluor® 488 Anti-HMGB1 antibody [EPR3507] (ab195010)
    • Alexa Fluor® 647 Anti-HMGB1 antibody [EPR3507] (ab195011)
    • HRP Anti-HMGB1 antibody [EPR3507] (ab195012)
    • Alexa Fluor® 594 Anti-HMGB1 antibody [EPR3507] (ab206894)
    • Alexa Fluor® 405 Anti-HMGB1 antibody [EPR3507] (ab206895)
    • Alexa Fluor® 555 Anti-HMGB1 antibody [EPR3507] (ab206896)
    • Anti-HMGB1 antibody [EPR3507] - BSA and Azide free (ab216986)
    • APC Anti-HMGB1 antibody [EPR3507] (ab225041)
    • PE Anti-HMGB1 antibody [EPR3507] (ab225042)
  • 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)
  • KO cell lines

    • Human HMGB1 knockout HeLa cell line (ab255395)
  • KO cell lysates

    • Human HMGB1 knockout HeLa cell lysate (ab263782)
  • Positive Controls

    • Hep G2 nuclear extract lysate (ab14660)
  • Recombinant Protein

    • Recombinant human HMGB1 protein (ab167718)
  • Related Products

    • Prestained Protein Ladder – Broad molecular weight (10-245 kDa) (ab116028)

应用

The Abpromise guarantee

Abpromise™承诺保证使用ab79823于以下的经测试应用

“应用说明”部分 下显示的仅为推荐的起始稀释度;实际最佳的稀释度/浓度应由使用者检定。

应用 Ab评论 说明
Flow Cyt (Intra)
1/30.

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

ICC/IF (1)
1/250.
WB (5)
1/10000 - 1/50000. Detects a band of approximately 25 kDa (predicted molecular weight: 25 kDa).
IHC-P (1)
1/350 - 1/400. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

说明
Flow Cyt (Intra)
1/30.

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

ICC/IF
1/250.
WB
1/10000 - 1/50000. Detects a band of approximately 25 kDa (predicted molecular weight: 25 kDa).
IHC-P
1/350 - 1/400. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

应用说明
Is unsuitable for IP.

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).
  • Target information above from: UniProt accession P09429 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • 数据库链接

    • Entrez Gene: 3146 Human
    • Entrez Gene: 100862258 Mouse
    • Entrez Gene: 15289 Mouse
    • Entrez Gene: 25459 Rat
    • Omim: 163905 Human
    • SwissProt: P09429 Human
    • SwissProt: P63158 Mouse
    • SwissProt: P63159 Rat
    • Unigene: 434102 Human
    • Unigene: 593339 Human
    • Unigene: 596078 Human
    • Unigene: 207047 Mouse
    • Unigene: 313345 Mouse
    • Unigene: 144565 Rat
    • Unigene: 15185 Rat
    • Unigene: 4121 Rat
    see all
  • 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

图片

  • Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823)
    Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823)
    All lanes : Anti-HMGB1 antibody [EPR3507] (ab79823) at 1/10000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : HMGB1 CRISPR/Cas9 edited HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 25 kDa
    Observed band size: 30 kDa why is the actual band size different from the predicted?



    Lanes 1- 2: Merged signal (red and green). Green - ab79823 observed at 30 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

     ab79823 was shown to react with HMGB1 in wild-type HeLa cells in western blot. Loss of signal at the expected size was observed when CRISPR/Cas9 edited cell line ab255395 (CRISPR/Cas9 edited cell lysate ab263782) was used. The band observed in lane 2 below 25kDa may represent truncated forms and cleaved fragments. Wild-type HeLa and HMGB1 CRISPR/Cas9 edited HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab79823 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  • Immunocytochemistry/ Immunofluorescence - Anti-HMGB1 antibody [EPR3507] (ab79823)
    Immunocytochemistry/ Immunofluorescence - Anti-HMGB1 antibody [EPR3507] (ab79823)

    ab79823 staining HMGB1 in wild-type HAP1 cells (top panel) and HMGB1 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde for 10 minutes, 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-Tween for 1 hour. The cells were then incubated with ab79823 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1 hour with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGB1 antibody [EPR3507] (ab79823)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGB1 antibody [EPR3507] (ab79823)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labeling HMGB1 with unpurified ab79823 at 1/350. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with Hematoxylin.

  • Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823)
    Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823)
    All lanes : Anti-HMGB1 antibody [EPR3507] (ab79823) at 1/10000 dilution

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : HMGB1 knockout HAP1 whole cell lysate
    Lane 3 : Jurkat whole cell lysate
    Lane 4 : HeLa whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 25 kDa



    Lanes 1 - 4: Merged signal (red and green). Green - ab79823 observed at 30 kDa. Red - loading control, ab9484, observed at 37 kDa.


    ab79823 was shown to specifically react with HMGB1 in wild-type HAP1 cells as signal was lost in HMGB1 knockout cells. Wild-type and HMGB1 knockout samples were subjected to SDS-PAGE. ab79823 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  • Immunocytochemistry/ Immunofluorescence - Anti-HMGB1 antibody [EPR3507] (ab79823)
    Immunocytochemistry/ Immunofluorescence - Anti-HMGB1 antibody [EPR3507] (ab79823)Image from Vicentino AR et al., PLoS One. 2012;7(6):e39104. Epub 2012 Jun 18. Fig 4.; doi:10.1371/journal.pone.0039104; June 18, 2012, PLoS ONE 7(6): e39104.

    Immunofluorescence analysis of murine RAW 264.7 macrophages, either untreated (upper panel) or treated with LPS (bottom panel). HMGB1 was stained using unpurified ab79823.
    Cells were fixed in paraformaldehyde, blocked in BSA for 1h, followed by permeabilization in 10% Triton X-100 for 30 min. Samples were incubated with primary antibody overnight at 4°C. An Alexa Fluor® 488-conjugated anti-rabbit IgG was used as the secondary antibody.

  • Immunocytochemistry/ Immunofluorescence - Anti-HMGB1 antibody [EPR3507] (ab79823)
    Immunocytochemistry/ Immunofluorescence - Anti-HMGB1 antibody [EPR3507] (ab79823)

    ICC/IF image of unpurified ab79823 stained DU 145 (Human prostate carcinoma cell line) cells. The cells were 4% formaldehyde fixed (10 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 ab79823 at 1/1000 dilution overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) 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.

  • Immunocytochemistry/ Immunofluorescence - Anti-HMGB1 antibody [EPR3507] (ab79823)
    Immunocytochemistry/ Immunofluorescence - Anti-HMGB1 antibody [EPR3507] (ab79823)

    Immunocytochemsitry/Immunofluorescence analysis of HeLa cells labelling HMGB1 (red) with unpurified ab79823 at 1/350. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor® 555-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody. Counterstained with DAPI (blue).

  • Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823)
    Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823)
    All lanes : Anti-HMGB1 antibody [EPR3507] (ab79823) at 1/10000 dilution (purified)

    Lane 1 : SK-BR-3 (Human mammary gland adenocarcinoma cell line) cell lysate
    Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

    Predicted band size: 25 kDa
    Observed band size: 25 kDa



    Blocking/Dilution buffer and concentration: 5% NFDM/TBST.

  • Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823)
    Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823)
    Anti-HMGB1 antibody [EPR3507] (ab79823) at 1/10000 dilution (purified) + Rat brain tissue lysate at 10 µg

    Secondary
    Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

    Predicted band size: 25 kDa
    Observed band size: 25 kDa



    Blocking/Dilution buffer and concentration: 5% NFDM/TBST.

  • Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823)
    Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823)
    All lanes : Anti-HMGB1 antibody [EPR3507] (ab79823) at 1/50000 dilution (unpurified)

    Lane 1 : SK-BR-3 cell lysate
    Lane 2 : HeLa cell lysate
    Lane 3 : HepG2 cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat anti-rabbit HRP conjugate at 1/2000 dilution

    Predicted band size: 25 kDa
    Observed band size: 25 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGB1 antibody [EPR3507] (ab79823)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGB1 antibody [EPR3507] (ab79823)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labeling HMGB1 with unpurified ab79823 at 1/250 dilution.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Flow Cytometry (Intracellular) - Anti-HMGB1 antibody [EPR3507] (ab79823)
    Flow Cytometry (Intracellular) - Anti-HMGB1 antibody [EPR3507] (ab79823)

    Overlay histogram showing HeLa (Human epithelial cell line from cervix adenocarcinoma) cells stained with unpurified ab79823 (red line). The cells were fixed with 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 (ab79823, 1/20 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (0.5µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in HeLa cells fixed with 4% paraformaldehyde/permeabilized with 0.1% PBS-Tween 20 used under the same conditions. 

     

     

  • Anti-HMGB1 antibody [EPR3507] (ab79823)
    Anti-HMGB1 antibody [EPR3507] (ab79823)

实验方案

  • Flow cytometry protocols
  • Immunohistochemistry protocols
  • Immunocytochemistry & immunofluorescence protocols
  • Western blot protocols

Click here to view the general protocols

数据表及文件

  • SDS download

  • Datasheet download

    Download

文献 (195)

发表研究结果有使用 ab79823?请让我们知道,以便我们可以引用本数据表中的参考文章。

ab79823 被引用在 195 文献中.

  • Xiao Y  et al. Relationship between the pyroptosis of fibroblast-like synoviocytes and HMGB1 secretion in knee osteoarthritis. Mol Med Rep 23:N/A (2021). PubMed: 33300062
  • Zhao H  et al. Propofol ameliorates endotoxin-induced myocardial cell injury by inhibiting inflammation and apoptosis via the PPAR?/HMGB1/NLRP3 axis. Mol Med Rep 23:N/A (2021). PubMed: 33398367
  • Kanai R  et al. Interferon-? enhances the therapeutic effect of mesenchymal stem cells on experimental renal fibrosis. Sci Rep 11:850 (2021). PubMed: 33441701
  • Dong H & Song J miR-142-3p reduces the viability of human cervical cancer cells by negatively regulating the cytoplasmic localization of HMGB1. Exp Ther Med 21:212 (2021). PubMed: 33500702
  • Lee JJ  et al. HMGB1 orchestrates STING-mediated senescence via TRIM30a modulation in cancer cells. Cell Death Discov 7:28 (2021). PubMed: 33558529
View all Publications for this product

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1-8 of 8 Abreviews or Q&A

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-HMGB1 antibody [EPR3507]

Excellent
Abreviews
Abreviews
abreview image
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (tonsil)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Leica ER2
Permeabilization
No
Specification
tonsil
Fixative
Formaldehyde
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

提交于 May 13 2021

Western blot abreview for Anti-HMGB1 antibody [EPR3507]

Excellent
Abreviews
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (fibroblast)
Gel Running Conditions
Non-reduced Non-Denaturing (Native) (gel 12%)
Loading amount
30 µg
Treatment
100uM Ethyl pyruvate
Specification
fibroblast
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · 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

提交于 Jul 07 2016

Immunocytochemistry/ Immunofluorescence abreview for Anti-HMGB1 antibody [EPR3507]

Good
Abreviews
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 25°C
Sample
Human Cell (Human prostate cancer cell line (PC-3))
Specification
Human prostate cancer cell line (PC-3)
Permeabilization
Yes - cooled acetone for 1' at -20C
Fixative
Methanol
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

提交于 Dec 31 2014

Western blot abreview for Anti-HMGB1 antibody [EPR3507]

Good
Abreviews
Abreviews
abreview image
Application
Western blot
Loading amount
25 µg
Gel Running Conditions
Reduced Denaturing
Sample
Human Cell lysate - whole cell (liver)
Specification
liver
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°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.

DR. Liyong Zhang

Verified customer

提交于 Dec 15 2014

Western blot abreview for Anti-HMGB1 antibody [EPR3507]

Good
Abreviews
Abreviews
abreview image
Application
Western blot
Loading amount
25 µg
Gel Running Conditions
Reduced Denaturing
Sample
Mouse Cell lysate - whole cell (hepatocytes)
Specification
hepatocytes
Treatment
20 ug/ml poly(I:C)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°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

提交于 Dec 01 2014

Western blot abreview for Anti-HMGB1 antibody [EPR3507]

Excellent
Abreviews
Abreviews
abreview image
Application
Western blot
Sample
Mouse Tissue lysate - whole (Liver)
Loading amount
10 µg
Specification
Liver
Gel Running Conditions
Reduced Non-Denaturing (Native) (4-20%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°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

提交于 Aug 29 2012

Western blot abreview for Anti-HMGB1 antibody [EPR3507]

Excellent
Abreviews
Abreviews
abreview image
Application
Western blot
Sample
Human Tissue lysate - whole (293 FT cell)
Loading amount
50 µg
Specification
293 FT cell
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°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

提交于 Sep 01 2010

Question

I would order it, if I order now, what would be its concentration (approximately)?

Regards

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Abcam community

Verified customer

Asked on May 16 2012

Answer

Thank you for your reply.

The concentration of this antibody is lot dependant, between 0.152 and 0.17 mg/mL.

I also would like to comment on the seemingly low concentration:

The IgG produced by rabbits have greater affinity and greater sensitivity than mouse IgG (about 100-1000 fold) and at the same time the total amount of IgG produced by the rabbit hybridomas is generally lower than in mouse hybridomas.

Many 'veteran' antibody users are generally familiar with the IgG amount provided from mouse antibodies and can judge how many reactions they may get or need to use based on concentration. Unfortunately, this experience is not transferable to rabbit monoclonal antibodies because the concentration scales are generally very different.

Since the specificity and sensitivity is much higher with rabbit monoclonal antibodies, less IgG is required per reaction.

I would like to reassure you that while the amount of IgG may not be comparable to mouse monoclonal antibodies, customers are receiving a comparable number of reactions/tests/blots/stainings per tube.

I hope this information is helpful. Please do not hesitate to contact me with any further questions in this matter.

Read More

Abcam Scientific Support

回复于 May 16 2012

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com

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