PRODUCT CODE: ET1601-2

Recombinant HMGB1 Monoclonal Antibody (ET1601-2)

  • Recombinant

Applications

  • WB

  • ICC

  • IF

  • IHC-P

  • FC

REACTIVITY

  • Human

  • Mouse

  • Rat

Western blot analysis of HMGB1 on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1601-2, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.<br />
 Positive control: <br />
 Lane 1: MCF-7 cell lysate<br />
 Lane 2: PC-12 cell lysate<br />
 Lane 3: F9 cell lysate
  • Western blot analysis of HMGB1 on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1601-2, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.<br />
 Positive control: <br />
 Lane 1: MCF-7 cell lysate<br />
 Lane 2: PC-12 cell lysate<br />
 Lane 3: F9 cell lysate
  • ICC staining of HMGB1 in NIH/3T3 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1601-2, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
  • ICC staining of HMGB1 in MCF-7 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1601-2, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
  • Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-HMGB1 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-2, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
  • Immunohistochemical analysis of paraffin-embedded human kidney tissue using anti-HMGB1 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-2, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
  • Immunohistochemical analysis of paraffin-embedded mouse brain tissue using anti-HMGB1 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-2, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
  • Immunohistochemical analysis of paraffin-embedded mouse kidney tissue using anti-HMGB1 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-2, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
  • Flow cytometric analysis of HMGB1 was done on PC-12 cells. The cells were fixed, permeabilized and stained with the primary antibody (ET1601-2, 1/50) (blue). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; red).
Western blot analysis of HMGB1 on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1601-2, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.
Positive control:
Lane 1: MCF-7 cell lysate
Lane 2: PC-12 cell lysate
Lane 3: F9 cell lysate

Applications

  • WB

  • ICC

  • IF

  • IHC-P

  • FC

REACTIVITY

  • Human

  • Mouse

  • Rat

SPECIFICATIONS

Product Type

Recombinant Rabbit monoclonal primary

Product Name

Recombinant HMGB1 Monoclonal Antibody (ET1601-2)

Immunogen

Synthetic peptide within human hmgb1 aa 160-190.

Host

Rabbit

Positive Control

MCF-7 cell lysate, PC-12 cell lysate, F9 cell lysate, NIH/3T3, MCF-7, human tonsil tissue, human kidney tissue, mouse brain tissue, mouse kidney tissue, PC-12.

Conjugation

Unconjugated

Clonality

Monoclonal

Clone Number

SA39-03

PROPERTIES

Form

Liquid

Storage Condition

Store at +4C after thawing. Aliquot store at -20C or -80C. Avoid repeated freeze / thaw cycles.

Storage Buffer

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Concentration

1 ug/ul

PURIFICATION

Protein A affinity purified.

MOLECULAR WEIGHT

25 kDa

Isotype

IgG

APPLICATION DILUTION

  • WB

  • 1:500-1:5,000

  • ICC/IF

  • 1:50-1:200

  • IHC-P

  • 1:50-1:200

  • FC

  • 1:50-1:100

TARGET

UNIPROT #

PROTEIN NAME

HMGB1

SYNONYMS

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

SEQUENCE SIMILARITIES

Belongs to the HMGB family.

TISSUE SPECIFICITY

Ubiquituous. Expressed in platelets.

POST-TRANSLATIONAL MODIFICATION

Phosphorylated at serine residues. Phosphorylation in both NLS regions is required for cytoplasmic translocation followed by secretion.; Acetylated on multiple sites upon stimulation with LPS. 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 (By similarity).; 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 (By similarity).; 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. Can be proteolytically cleaved by a thrombin:thrombomodulin complex; reduces binding to heparin and proinflammatory activities (By similarity).

SUBCELLULAR LOCATION

Cytoplasm, Nucleus, Cell membrane, Secreted, Chromosome

FUNCTION

Like the histones, HMGB1, also known as high-mobility group protein 1 (HMG-1) is among the most important chromatin proteins. In the nucleus HMGB1 interacts with nucleosomes, transcription factors, and histones. This nuclear protein organizes the DNA and regulates transcription. After binding, HMGB1 bends DNA, which facilitates the binding of other proteins. HMGB1 is secreted by immune cells (like macrophages, monocytes and dendritic cells) through leaderless secretory pathway. Activated macrophages and monocytes secrete HMGB1 as a cytokine mediator of Inflammation. In recent research, HMGB1 has been reported as a novel biomarker for human ovarian cancer