Key features and details
- Rabbit polyclonal to HMGB1 - N-terminal
- Suitable for: WB, IP, IHC-P, IHC-Fr, ICC/IF
- Reacts with: Mouse, Rat, Human
- Isotype: IgG
Product nameAnti-HMGB1 antibody - N-terminal
See all HMGB1 primary antibodies
DescriptionRabbit polyclonal to HMGB1 - N-terminal
Tested applicationsSuitable for: WB, IP, IHC-P, IHC-Fr, ICC/IFmore details
Species reactivityReacts with: Mouse, Rat, Human
Predicted to work with: Rabbit, Chicken, Cow, Dog, Pig, Xenopus laevis, Rhesus monkey
Synthetic peptide within Human HMGB1 (N terminal). The exact sequence is proprietary. Carrier-protein conjugated.
Database link: P09429
- WB: HEK-293T, A431, HeLa and HepG2 whole cell extracts; Mouse and rat brain tissue extracts. IHC-P: SkHep1 xenograft tissue; mouse urinary bladder and vein tissues. IHC-Fr: Adult mouse cerebellum tissue. ICC/IF: NTERA-2 cl.D1 [NT2/D1] cells; DIV9 rat E18 primary cortical neurons. IP: HEK-293T whole cell extract.
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In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.
We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.
Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.
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Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
Storage bufferpH: 7.00
Preservative: 0.01% Thimerosal (merthiolate)
Constituents: 1.21% Tris, 0.75% Glycine, 20% Glycerol (glycerin, glycerine)
Concentration information loading...
PurityImmunogen affinity purified
- HeLa whole cell lysate (ab150035)
- HepG2 whole cell lysate (ab166833)
- HeLa whole cell lysate (ab29545)
- A431 whole cell lysate (ab30132)
- Mouse brain tissue lysate - total protein (ab30151)
- Mouse brain tissue lysate - total protein (ab4022)
- A431 whole cell lysate (ab7909)
- 293T whole cell lysate (ab95494)
Our Abpromise guarantee covers the use of ab228624 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/500 - 1/10000. Predicted molecular weight: 25 kDa.|
|IP||1/100 - 1/500.|
|IHC-P||1/100 - 1/1000.|
|IHC-Fr||1/100 - 1/1000.|
|ICC/IF||1/100 - 1/1000.|
FunctionMultifunctional 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.
Tissue specificityUbiquituous. Expressed in platelets (PubMed:11154118).
Sequence similaritiesBelongs to the HMGB family.
Contains 2 HMG box DNA-binding domains.
DomainHMG 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).
modificationsPhosphorylated 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.
Cellular localizationNucleus. 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
- Amphoterin antibody
- Chromosomal protein, nonhistone, HMG1 antibody
- DKFZp686A04236 antibody
All lanes : Anti-HMGB1 antibody - N-terminal (ab228624) at 1/5000 dilution
Lane 1 : Non-transfected HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell extract
Lane 2 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with HMGB1 shRNA, whole cell extract
Lysates/proteins at 30 µg per lane.
Predicted band size: 25 kDa
12% SDS-PAGE gel.
All lanes : Anti-HMGB1 antibody - N-terminal (ab228624) at 1/1000 dilution
Lane 1 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell extract
Lane 2 : A431 (human epidermoid carcinoma cell line) whole cell extract
Lane 3 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell extracts
Lane 4 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell extract
Lysates/proteins at 30 µg per lane.
Predicted band size: 25 kDa
12% SDS-PAGE gel.
Anti-HMGB1 antibody - N-terminal (ab228624) at 1/500 dilution + Mouse brain tissue extract at 50 µg
Predicted band size: 25 kDa
12% SDS-PAGE gel.
Anti-HMGB1 antibody - N-terminal (ab228624) at 1/500 dilution + Rat brain tissue extract at 50 µg
Predicted band size: 25 kDa
12% SDS-PAGE gel.
Frozen sections of adult mouse cerebellum tissue stained for HMGB1 (green) using ab228624 at 1/250 dilution in immunohistochemical analysis.
Nuclear counterstain: Fluoroshield with DAPI (blue). NF-H, stained by an anti-NF-H antibody (red) at 1/500 dilution.
Paraffin-embedded SkHep1 (human) xenograft tissue stained for HMGB1 using ab228624 at 1/500 dilution in immunohistochemical analysis.
Paraffin-embedded mouse urinary bladder tissue stained for HMGB1 using ab228624 at 1/500 dilution in immunohistochemical analysis.
Paraffin-embedded mouse vein tissue stained for HMGB1 using ab228624 at 1/500 dilution in immunohistochemical analysis.
4% paraformaldehyde-fixed NTERA-2 cl.D1 [NT2/D1] (human malignant pluripotent embryonic carcinoma cell line) cells stained for HMGB1 (green) using ab228624 at 1/500 dilution in ICC/IF.
Nuclear counterstain: Hoechst 33342 (blue).
4% paraformaldehyde-fixed DIV9 rat E18 primary cortical neurons stained for HMGB1 (green) using ab228624 at 1/500 dilution in ICC/IF.
Counterstain: beta Tubulin 3/Tuj1, a neuron cell marker, stained by an anti-beta Tubulin 3/Tuj1 antibody at 1/500 dilution (red).
HMGB1 was immunoprecipitated from HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell extract with 5 µg of ab228624. Western blot was performed from the immunoprecipitate using ab228624. Anti-Rabbit IgG was used as a secondary reagent.
Lane 1: Control IgG instead of ab228624 in HEK-293T whole cell extract.
Lane 2: ab228624 IP in HEK-293T whole cell extract.
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
ab228624 has not yet been referenced specifically in any publications.