HMGB1/HMG-1 Antibody - BSA Free

Western Blot: HMGB1/HMG-1 Antibody [NB100-2322] - Western blot shows lysates of HEK293T human embryonic kidney parental cell line and HMGB1 knockout (KO) HEK293T cell line. PVDF membrane was probed with 1.0 ug/ml of Rabbit Anti-Human HMGB1 Polyclonal Antibody (Catalog # NB100-2322) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog #HAF008). Specific band was detected for HMGB1 at approximately 30 kDa (as indicated) in the parental HEK293T cell line, but is not detectable in the knockout HEK293T cell line. This experiment was conducted under reducing conditions.

Western Blot: HMGB1/HMG-1 Antibody [NB100-2322] - Total protein from SHSY-5Y, MCF7, Neuro2A and HeLa was separated on a 12% gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 2.0 ug/mL anti-HMGB1 in 1% non-fat milk in TBST and detected with an anti-rabbit HRP secondary antibody using chemiluminescence.

Immunohistochemistry-Paraffin: HMGB1/HMG-1 Antibody [NB100-2322] - Staining of HMGB1 in mouse liver using NB100-2322.

Simple Western: HMGB1/HMG-1 Antibody [NB100-2322] - Image shows a specific band for HMGB1 in 0.05 mg/mL of Jurkat lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.

Immunocytochemistry/Immunofluorescence: HMGB1/HMG-1 Antibody [NB100-2322] - MCF7 cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.5% Triton X-100 in PBS for 5 minutes. The cells were incubated with anti-HMGB1/HMG-1 Antibody NB100-2322 at 1 ug/ml for overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:1000 dilution for 60 minutes. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.

Immunocytochemistry/Immunofluorescence: HMGB1/HMG-1 Antibody [NB100-2322] - Neuro2a cells were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton-X100. The cells were incubated with anti-HMGB1 at 5 ug/ml overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:500 dilution. Alpha tubulin (DM1A) NB100-690 was used as a co-stain at a 1:1000 dilution and detected with an anti-mouse Dylight 550 (Red) at a 1:500 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.

Immunohistochemistry-Paraffin: HMGB1/HMG-1 Antibody [NB100-2322] - Rabbit blood vessel. Image from verified customer review.

Western Blot: HMGB1/HMG-1 Antibody [NB100-2322] - The onset of innate immune response after I/R injury was compared in wt and PT-SSAT-Cko animals. Time course of the expression of HMGB1, TLR2 and 4 were compared in the kidneys of sham-operated and injured wt and PT-SSAT-Cko mice. The data are representative of three independent experiments. Image collected and cropped by CiteAb from the following publication (//doi.org/10.1371/journal.pone.0110161) licensed under a CC-BY license.

Immunocytochemistry/Immunofluorescence: HMGB1/HMG-1 Antibody [NB100-2322] - NIH3T3 cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.5% Triton X-100 in PBS for 5 minutes. The cells were incubated with anti-HMGB1/HMG-1 Antibody NB100-2322 at 1 ug/ml for overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:1000 dilution for 60 minutes. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.

Flow Cytometry: HMGB1/HMG-1 Antibody [NB100-2322] - An intracellular stain was performed on HeLa cells with HMGB1/HMG-1 Antibody NB100-2322AF488 (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 5 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to Alexa Fluor 488.

Western Blot: HMGB1/HMG-1 Antibody [NB100-2322] - Hepatocyte protein lysate at 1:1000 4C overnight. Image from verfified customer review.

Immunocytochemistry/Immunofluorescence: HMGB1/HMG-1 Antibody [NB100-2322] - HeLa cells were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton-X100. The cells were incubated with anti-HMGB1 [NB100-2322] at a 1:200 dilution overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:500 dilution. Alpha tubulin (DM1A) [NB100-690] was used as a co-stain at a 1:1000 dilution and detected with an anti-mouse Dylight 550 (Red) at a 1:500 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.

Flow (Intracellular): HMGB1/HMG-1 Antibody [NB100-2322] - An intracellular stain was performed on HeLa with NB100-2322 and a matched isotype control. Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 1 ug/mL for 30 minutes at room temperature, followed by Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody.

Flow Cytometry: HMGB1/HMG-1 Antibody [NB100-2322] - An intracellular stain was performed on RH-30 cells with NB100-2322F (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 10 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to FITC.

ELISA: HMGB1/HMG-1 Antibody [NB100-2322] - A dose-dependent titration of the HRP conjugated anti-HMGB1 antibody on recombinant human HMGB1 protein. Image from verified customer review. Image using the HRP format of this antibody.

Expression of endogenous ligands for TLR2. (A) Expression of biglycan in AD-TLR2KO mice increased significantly compared with that in WT, AD, and TLR2KO mice (p0.05).

Western Blot: HMGB1/HMG-1 Antibody [NB100-2322] - Expression of endogenous ligands for TLR2. (A) Expression of biglycan in AD-TLR2KO mice increased significantly compared with that in WT, AD, & TLR2KO mice (p0.05). Image collected & cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31509519), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

• Reactivity: Hu, Mu, Rt, Bv, Ca, Rb, Sh, Po, Eq
• Applications: WB, Simple Western, ELISA, Flow, ICC/IF, IHC, KD
• Clonality: Polyclonal
• Host: Rabbit
• Conjugate: Unconjugated
• Format: BSA Free
• Concentration: 1 mg/ml

HMGB1/HMG-1 Antibody - BSA Free Summary

• Immunogen: A synthetic peptide made to an internal portion of the human HMGB1 protein sequence (between residues 100-200). [UniProt #P09429]
• Localization: Nuclear
• Predicted Species: Porcine (100%), Equine (100%). Backed by our 100% Guarantee.
• Isotype: IgG
• Clonality: Polyclonal
• Host: Rabbit
• Gene: HMGB1
• Purity: Immunogen affinity purified

Applications/Dilutions

• Dilutions:
  • ELISA
  • Flow (Intracellular)
  • Flow Cytometry 1:100
  • Immunocytochemistry/ Immunofluorescence 0.05 ug/ml
  • Immunohistochemistry 1:100-1:250
  • Immunohistochemistry-Paraffin 1:100-1:250
  • Knockdown Validated
  • Simple Western 1:2000
  • Western Blot 0.5-1.0 ug/ml
• Application Notes: In Western blot a band is seen approx. 29 kDa. It has been reported that the HRP conjugated format (Catalog# NB100-2322H) of this antibody works well for use in ELISA.
  
  In Simple Western only 10 - 15 uL of the recommended dilution is used per data point.
  See Simple Western Antibody Database for Simple Western validation: Tested in Jurkat lysate 0.05 mg/mL, separated by Size, antibody dilution of 1:2000. Separated by Size-Wes, Sally Sue/Peggy Sue.
  The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• Theoretical MW: 29 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• Control:

  HMGB1/HMG-1 Overexpression Lysate
  HeLa Whole Cell Lysate

• Control Peptide:

  HMGB1/HMG-1 Peptide (NB100-2322PEP)

Reactivity Notes

Rabbit reactivity reported in scientific literature (PMID: 27401639).

Packaging, Storage & Formulations

• Storage: Store at 4C. Do not freeze.
• Buffer: PBS
• Preservative: 0.02% Sodium Azide
• Concentration: 1 mg/ml
• Purity: Immunogen affinity purified

Alternate Names for HMGB1/HMG-1 Antibody - BSA Free

• Amphoterin
• high mobility group box 1
• High mobility group protein 1
• high mobility group protein B1
• high-mobility group (nonhistone chromosomal) protein 1
• high-mobility group box 1
• HMG1
• HMG-1
• HMG1DKFZp686A04236
• HMG3
• HMGB1
• SBP-1
• Sulfoglucuronyl carbohydrate binding protein

Background

HMGB1 (high mobility group protein B1) is a member of HMGB family and it has been extensively studied as a transcription/growth factor that is most ubiquitous and evolutionarily conserved protein in eukaryotic organisms. HMGB1 is localized in nucleus, however gets translocated to cytoplasm or may get secreted also. It is a DNA binding protein that preferentially binds with ssDNA and contains 2 HMG box DNA-binding domains with ability of chromatin association for bending DNA. HMGB1 implicates in in V(D)J recombination by acting as a cofactor of RAG complex that contains core components RAG1 and RAG2, as well as an associated component HMGB1 or HMGB2. HMGB1 acts by stimulating cleavage and RAG protein binding at 23bp spacer of conserved RSS (recombination signal sequences). Originally described as a nonhistone nuclear protein with transcriptional regulatory properties, HMGB1 (especially the cytoplasmic/secreted form) has also now been recognized as a late mediator in septic shock as well as a pro-inflammatory cytokine. It gets translocated to cytoplasm or secreted when cells get exposed to inflammatory mediators (LPS, TNF-alpha, IFN-gamma) or via passive process during cellular necrosis. HMGB1 has been implicated in the pathogenesis of various inflammatory and autoimmune diseases, such as SLE, RA and ankylosing spondylitis.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for HMGB1/HMG-1 Antibody (NB100-2322)(36)

Publications using NB100-2322

• Dalvi S, Roll M, Chatterjee A, Kumar LK et Al. Human iPSC-based disease modeling studies identify a common mechanistic defect and potential therapies for AMD and related macular dystrophies Dev Cell 2024-10-03 [PMID: 39362220]
• Abbaszadeh F, Jorjani M, Joghataei MT et al. Astaxanthin ameliorates spinal cord edema and astrocyte activation via suppression of HMGB1/TLR4/NF-?B signaling pathway in a rat model of spinal cord injury Naunyn-Schmiedeberg's archives of pharmacology 2023-05-05 [PMID: 37145127] (WB, Rat)
• Lin T, Sheng L, Wan L et al. Suppressing HMGB1-TLR4-Mediated Neuroinflammation Alleviates Morphine Tolerance via Inhibiting AMPK-HO-1 Pathway in The Spinal Cord of Mice Research Square 2020-12-16 (IF/IHC, WB, Mouse)
• Vilca SJ, Margetts AV, Höglund L et Al. Microglia contribute to methamphetamine reinforcement and reflect persistent transcriptional and morphological adaptations to the drug Brain Behav Immun 2024-07-25 [PMID: 38838836]
• Shih-Hong Khoo, Pei-Ru Wu, Kun-Tu Yeh, Shih-Lan Hsu, Chi-Hao Wu Biological and clinical significance of the AGE-RAGE axis in the aggressiveness and prognosis of prostate cancer Journal of Food and Drug Analysis 2023-01-01 [PMID: 38526823]
• Iram Zafar, Shajer Manzoor, Nithya Mariappan, Shama Ahmad, Mohammad Athar, Veena Antony, Aftab Ahmad A Murine Model of Vesicant-Induced Acute Lung Injury. The Journal of pharmacology and experimental therapeutics 2024-01-19 [PMID: 38050084]
• Yang W, Hong SA, Kim JM et al. The immunologic phenotype of thrombi is associated with future vascular events after cerebral infarction Journal of neurointerventional surgery 2023-05-17 [PMID: 37197936]
• Hao Y, Gu Z, Yu Z et al. Photodynamic Therapy in Combination with the Hepatitis B Core Virus-like Particles (HBc VLPs) to Prime Anticancer Immunity for Colorectal Cancer Treatment Cancers 2022-05-31 [PMID: 35681703] (FLOW, Mouse)
• Ershova ES, Shmarina GV, Porokhovnik LN Et al. In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls Genes (Basel) 2022-03-25 [PMID: 35328103] (FLOW, Human)
  
  Details:
  Citation using the FITC version of this antibody.
• Montes de Oca R, Alavi AS, Vitali N et al. Belantamab Mafodotin (GSK2857916) Drives Immunogenic Cell Death and Immune-mediated Antitumor Responses In Vivo Molecular Cancer Therapeutics 2021-10-01 [PMID: 34253590] (Flow Cytometry)
• Baker BC, Heazell AEP, Sibley C et al. Hypoxia and oxidative stress induce sterile placental inflammation in vitro Scientific reports 2021-03-31 [PMID: 33790316] (IF/IHC, Human)
• Ghaffari, S, Jang, E Et al. Endothelial HMGB1 Is a Critical Regulator of LDL Transcytosis via an SREBP2-SR-BI Axis. Arterioscler Thromb Vasc Biol 2021-01-01 [PMID: 33054399] (IF/IHC, Mouse)
• Jella KK, Nasti TH, Li Z et al. Exosome-containing preparations from post-irradiated mouse melanoma cells delay melanoma growth in vivo by a natural killer cell-dependent mechanism Int. J. Radiat. Oncol. Biol. Phys. 2020-06-16 [PMID: 32561502]
• Manzoor, S, Mariappan, N Et al. Cutaneous lewisite exposure causes acute lung injury. Ann N Y Acad Sci 2020-11-01 [PMID: 32329907] (IF/IHC, Mouse)
• Evran S, Calis F, Akkaya E et al. The effect of high mobility group box-1 protein on cerebral edema, blood-brain barrier, oxidative stress and apoptosis in an experimental traumatic brain injury model Brain Res. Bull. 2019-11-09 [PMID: 31715313] (IF/IHC, Rat)
• Domingo-Vidal M, Whitaker-Menezes D, Martos-Rus C et al. Cigarette Smoke Induces Metabolic Reprogramming of the Tumor Stroma in Head and Neck Squamous Cell Carcinoma Mol. Cancer Res. 2019-06-25 [PMID: 31239287] (ICC/IF, Rat)
• Silvia Ezquerro, Fátima Mocha, Gema Frühbeck, Rocío Guzmán-Ruiz, Víctor Valentí, Carmen Mugueta, Sara Becerril, Victoria Catalán, Javier Gómez-Ambrosi, Camilo Silva, Javier Salvador, Inmaculada Colina, María M Malagón, Amaia Rodríguez Ghrelin Reduces TNF-alpha -Induced Human Hepatocyte Apoptosis, Autophagy, and Pyroptosis: Role in Obesity-Associated NAFLD. The Journal of clinical endocrinology and metabolism 2019-12-03 [PMID: 30137403]
• Li T, Liu B, Guan H et al. PGE2 increases inflammatory damage in Escherichia coli-infected bovine endometrial tissue in vitro via the EP4-PKA signaling pathway. Biol. Reprod. 2018-07-11 [PMID: 30010723]
• Gao R, Ma J, Wen Z et al. Tumor cell-released autophagosomes (TRAP) enhance apoptosis and immunosuppressive functions of neutrophils Oncoimmunology. 2018-03-06 [PMID: 29872581] (WB, Mouse, Human)
  
  Details:
  Citation used the Alexa Fluor 488 format of this antibody.
• Kim SY, Son M, Lee SE et al. High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation. Front Immunol 2018-04-11 [PMID: 29696019] (WB, Mouse)
• Xiao J, Yao J, Jia L et al. Protective Effect of Met12, a Small Peptide Inhibitor of Fas, on the Retinal Pigment Epithelium and Photoreceptor After Sodium Iodate Injury. Invest. Ophthalmol. Vis. Sci. 2017-03-01 [PMID: 28346613]
• Coleman LG, Zou J, Crews FT Microglial-derived miRNA let-7 and HMGB1 contribute to ethanol-induced neurotoxicity via TLR7 J Neuroinflammation Jan 25 2017 [PMID: 28118842] (FLOW, ELISA, Human) J Neuroinflammation. 2017-01-25 [PMID: 28118842] (ELISA, FLOW, Human)
  
  Details:
  Citation using the Alexa Fluor 488 form of this antibody.
• Zhou M, Wen Z, Cheng F et al. Tumor-released autophagosomes induce IL-10-producing B cells with suppressive activity on T lymphocytes via TLR2-MyD88-NF-kappa B signal pathway Oncoimmunology. 2016-05-12 [PMID: 27622036] (WB, Human)
• Kim JS, Lee SG, Oh J et al. Development of Advanced Atherosclerotic Plaque by Injection of Inflammatory Proteins in a Rabbit Iliac Artery Model Yonsei Med. J. 2016-09-01 [PMID: 27401639] (IF/IHC, Rabbit)
• Feng Y, Yang S, Ma Y et al. Role of Toll-like receptors in diabetic renal lesions in a miniature pig model. Sci Adv 2015-06-01 [PMID: 26601192] (IF/IHC, Bovine)
• Zahedi K, Barone S, Wang Y et al. Proximal tubule epithelial cell specific ablation of the spermidine/spermine n1-acetyltransferase gene reduces the severity of renal ischemia/reperfusion injury. PLoS OnE. 2014-11-13 [PMID: 25390069] (WB, Human)
• Chi-Cheng Lu, Bo-Ru Huang, Pei-Jyuan Liao, Gow-Chin Yen. Ursolic acid triggers nonprogrammed death (necrosis) in human glioblastoma multiforme DBTRG-05MG cells through MPT pore opening and ATP decline. Mol Nutr Food Res. 2014-11-01 [PMID: 25131308]
• Robinson AP, Caldis MW, Harp CT et al. High-mobility group box 1 protein (HMGB1) neutralization ameliorates experimental autoimmune encephalomyelitis. J Autoimmun 2013-01-01 [PMID: 23514872] (ICC/IF, Mouse)
• Kang R, Tang D, Schapiro NE et al. The HMGB1/RAGE inflammatory pathway promotes pancreatic tumor growth by regulating mitochondrial bioenergetics. Oncogene 2013-01-14 [PMID: 23318458] (WB, Mouse)
• Montie HL, Pestell RG, Merry DE. SIRT1 Modulates Aggregation and Toxicity through Deacetylation of the Androgen Receptor in Cell Models of SBMA. J Neurosci 3031(48):17425-17436. 2011-11-01 [PMID: 22131404]
• Tamura Yoshiaki, Chiba Yuko, Tanioka Toshihiro et al. NO donor induces Nec-1-inhibitable, but RIP1-independent, necrotic cell death in pancreatic B-cells. Febs Letters 2011-01-01 [PMID: 21888904] (WB, Mouse)
• Tang D, Kang R, Livesey KM et al. High-mobility group box 1 is essential for mitochondrial quality control Cell Metab 2011-06-01 [PMID: 21641551] (ICC/IF, WB, Mouse)
• Fazio EN, Dimattia GE, Chadi S et al. Stanniocalcin 2 alters PERK signalling and reduces cellular injury during cerulein induced pancreatitis in mice BMC Cell Biol 2011-05-05 [PMID: 21545732] (IF/IHC, ICC/IF, Mouse)
• Jiang Y, Scott KL, Kwak SJ et al. Sds22/PP1 links epithelial integrity and tumor suppression via regulation of myosin II and JNK signaling. Oncogene 2011-07-21 [PMID: 21399659]
• Barlan AU, Griffin TM, McGuire KA, Wiethoff CM. Adenovirus membrane penetration activates the NLRP3 inflammasome. J Virol. 2010-10-27 [PMID: 20980503] (WB, Human)

Reviews for HMGB1/HMG-1 Antibody (NB100-2322) (5)

Average Rating: 4.4

(Based on 5 reviews)

We have 5 reviews tested in 4 species: Human, Mouse, Rat, Rabbit.

reviewed by: seulgee lee IHC-P Rabbit 04/24/2018

Summary

• Application: Immunohistochemistry-Paraffin
• Sample Tested: veesel
• Species: Rabbit
• Lot: D-1

reviewed by: Verified Customer WB Rat 03/28/2018

Summary

• Application: Western Blot
• Sample Tested: Brain homogenate
• Species: Rat
• Lot: Q-1

Comments

• Comments: Blocked in 5% milk + 1% BSA in TBS for 2 h, incubated overnight in 1:500 HMGB1 antibody at 4 degrees C and then 2 h at room temperature with HRP-conjugated secondary.

reviewed by: Liyong Zhang WB Human 11/25/2017

Summary

• Application: Western Blot
• Sample Tested: mouse hepatocytes and HepG2 cells
• Species: Human
• Lot: F-1

reviewed by: Verified Customer Simple Western Mouse 12/30/2015

Summary

• Application: Simple Western
• Sample Tested: mouse serum
• Species: Mouse

reviewed by: Liyong Zhang WB Mouse 06/26/2014

Summary

• Application: Western Blot
• Species: Mouse

FAQs for HMGB1/HMG-1 Antibody (NB100-2322). (Showing 1 - 1 of 1 FAQs).

1. I'd like to use NB100-2322 for FACS. One of the images shows successful staining. Which permeabilization method and fixative was used? I've seen conflicting reports, especially for fixation.
   • This image was obtained using 10% formalin as fixative and 90% methanol for permeabilization.

Bioinformatics

• Gene Symbol: HMGB1