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HMGB1 Recombinant Rabbit Monoclonal Antibody [SA39-03]

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Catalog# ET1601-2

HMGB1 Recombinant Rabbit Monoclonal Antibody [SA39-03]

Application

  • WB

  • IF-Cell

  • IF-Tissue

  • IHC-P

  • FC

  • IHC-Fr

Reactivity

  • Human

  • Mouse

  • Rat

BSA and Azide free
Conjugation

  • unconjugated

This product has been cited in peer reviewed publications, see list HERE

Overview

Product Name

HMGB1 Recombinant Rabbit Monoclonal Antibody [SA39-03]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic peptide within Human HMGB1 aa 151-200 / 215.

Species Reactivity

Human, Mouse, Rat

Validated Applications

WB, IF-Cell, IF-Tissue, IHC-P, FC, IHC-Fr

Molecular Weight

Predicted band size: 25 kDa

Positive Control

HeLa, HepG2 cell lysate, HeLa cell lysate, HCT 116 cell lysate, A549 cell lysate, Jurkat cell lysate, C2C12 cell lysate, C6 cell lysate, Mouse thymus tissue lysate, Rat spleen tissue lysate, MCF-7, human kidney tissue, mouse brain tissue, mouse hippocampus tissue, rat brain tissue, rat hippocampus tissue, C2C12, C6, HCT 116.

Conjugation

unconjugated

Clone Number

SA39-03

RRID

AB_3069602

Product Features

Form

Liquid

Concentration

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.

Storage Buffer

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

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:20,000-1:50,000

  • IF-Cell

  • 1:500

  • IF-Tissue

  • 1:2,000

  • IHC-P

  • 1:5,000-1:20,000

  • FC

  • 1:1,000

  • IHC-Fr

  • 1:500

Target

Function

High mobility group box 1 protein, also known as high-mobility group protein 1 (HMG-1) and amphoterin, is a protein that in humans is encoded by the HMGB1 gene. HMG-1 belongs to the high mobility group and contains a HMG-box domain. Like the histones, HMGB1 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 supports transcription of many genes in interactions with many transcription factors. It also interacts with nucleosomes to loosen packed DNA and remodel the chromatin. Contact with core histones changes the structure of nucleosomes. The presence of HMGB1 in the nucleus depends on posttranslational modifications. When the protein is not acetylated, it stays in the nucleus, but hyperacetylation on lysine residues causes it to translocate into the cytosol. HMGB1 has been shown to play an important role in helping the RAG endonuclease form a paired complex during V(D)J recombination.

Background References

1. "Novel role of PKR in inflammasome activation and HMGB1 release." Lu B., Nakamura T., Inouye K., Li J., Tang Y., Lundbaeck P., Valdes-Ferrer S.I., Olofsson P.S., Kalb T., Roth J., Zou Y., Erlandsson-Harris H., Yang H., Ting J.P., Wang H., Andersson U., Antoine D.J., Chavan S.S., Hotamisligil G.S., Tracey K.J. Nature 488:670-674(2012).

2. "The genetic variation of the human HMGB1 gene." Kornblit B., Munthe-Fog L., Petersen S., Madsen H., Vindeloev L., Garred P. Tissue Antigens 70:151-156(2007).

Sequence Similarity

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

UNIPROT

SwissProt: P09429 Human

SwissProt: P63158 Mouse

SwissProt: P63159 Rat

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

Expand

Images

  • Western blot analysis of HMGB1 on different lysates with Rabbit anti-HMGB1 antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/50,000 dilution and competitor's antibody at 1/10,000 dilution.<br /><br />Lane 1: HepG2 cell lysate<br />Lane 2: HeLa cell lysate<br />Lane 3: HCT 116 cell lysate<br />Lane 4: A549 cell lysate<br />Lane 5: Jurkat cell lysate<br />Lane 6: C2C12 cell lysate<br />Lane 7: C6 cell lysate<br /><br />Lysates/proteins at 15 µg/Lane.<br /><br />Predicted band size: 25 kDa<br />Observed band size: 25 kDa<br /><br />Exposure time: 21 seconds; ECL: K1801;<br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/50,000 dilution and competitor's antibody at 1/10,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of HMGB1 on different lysates with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/50,000 dilution and competitor's antibody at 1/10,000 dilution.

    Lane 1: HepG2 cell lysate
    Lane 2: HeLa cell lysate
    Lane 3: HCT 116 cell lysate
    Lane 4: A549 cell lysate
    Lane 5: Jurkat cell lysate
    Lane 6: C2C12 cell lysate
    Lane 7: C6 cell lysate

    Lysates/proteins at 15 µg/Lane.

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

    Exposure time: 21 seconds; ECL: K1801;
    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1601-2) at 1/50,000 dilution and competitor's antibody at 1/10,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • <span style="font-weight: bold;">☑ Knockout (KO)</span><br /><br />Western blot analysis of HMGB1 with anti-HMGB1 antibody [SA39-03] (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/50,000 dilution.<br /><br />Lane 1: Wild-type Raw264.7 whole cell lysate.<br />Lane 2: HMGB1 knockout Raw264.7 whole cell lysate.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary Anti-HMGB1 antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>, 1/50,000) and Anti-HSP90 antibody (<a href="/products/ET1605-56" style="font-weight: bold;text-decoration: underline;">ET1605-56</a>, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.<br /><br />Cell lysate was provided by Ubigene Biosciences (Ubigene Biosciences Co., Ltd., Guangzhou, China).

    ☑ Knockout (KO)

    Western blot analysis of HMGB1 with anti-HMGB1 antibody [SA39-03] (ET1601-2) at 1/50,000 dilution.

    Lane 1: Wild-type Raw264.7 whole cell lysate.
    Lane 2: HMGB1 knockout Raw264.7 whole cell lysate.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary Anti-HMGB1 antibody (ET1601-2, 1/50,000) and Anti-HSP90 antibody (ET1605-56, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

    Cell lysate was provided by Ubigene Biosciences (Ubigene Biosciences Co., Ltd., Guangzhou, China).

  • Western blot analysis of HMGB1 on different lysates with Rabbit anti-HMGB1 antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/20,000 dilution.<br /><br />Lane 1: HeLa cell lysate (20 µg/Lane)<br />Lane 2: HCT 116 cell lysate (20 µg/Lane)<br />Lane 3: A549 cell lysate (20 µg/Lane)<br />Lane 4: HepG2 cell lysate (20 µg/Lane)<br />Lane 5: Jurkat cell lysate (20 µg/Lane)<br />Lane 6: Mouse thymus tissue lysate (20 µg/Lane)<br />Lane 7: Rat spleen tissue lysate (30 µg/Lane)<br /><br />Predicted band size: 25 kDa<br />Observed band size: 25 kDa<br /><br />Exposure time: 3 minutes 10 seconds; ECL: K1801;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/20,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of HMGB1 on different lysates with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/20,000 dilution.

    Lane 1: HeLa cell lysate (20 µg/Lane)
    Lane 2: HCT 116 cell lysate (20 µg/Lane)
    Lane 3: A549 cell lysate (20 µg/Lane)
    Lane 4: HepG2 cell lysate (20 µg/Lane)
    Lane 5: Jurkat cell lysate (20 µg/Lane)
    Lane 6: Mouse thymus tissue lysate (20 µg/Lane)
    Lane 7: Rat spleen tissue lysate (30 µg/Lane)

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

    Exposure time: 3 minutes 10 seconds; ECL: K1801;

    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1601-2) at 1/20,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • Immunocytochemistry analysis of HeLa cells labeling HMGB1 with Rabbit anti-HMGB1 antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/500 dilution and competitor's antibody at 1/250 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-HMGB1 antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/500 dilution and competitor's antibody at 1/250 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.<br /><br />Beta tubulin (<a href="/products/M1305-2" style="font-weight: bold;text-decoration: underline;">M1305-2</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of HeLa cells labeling HMGB1 with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/500 dilution and competitor's antibody at 1/250 dilution.

    Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/500 dilution and competitor's antibody at 1/250 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

  • Immunocytochemistry analysis of MCF-7 cells labeling HMGB1 with Rabbit anti-HMGB1 antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/100 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-HMGB1 antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/100 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    Immunocytochemistry analysis of MCF-7 cells labeling HMGB1 with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/100 dilution.

    Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/100 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

  • Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-HMGB1 antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/20,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/20,000 dilution for 1 hour 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 with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/20,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-2) at 1/20,000 dilution for 1 hour 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 with Rabbit anti-HMGB1 antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/20,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/20,000 dilution for 1 hour 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 with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/20,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-2) at 1/20,000 dilution for 1 hour 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 hippocampus tissue with Rabbit anti-HMGB1 antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/20,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/20,000 dilution for 1 hour 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 hippocampus tissue with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/20,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-2) at 1/20,000 dilution for 1 hour 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 rat brain tissue with Rabbit anti-HMGB1 antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/20,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/20,000 dilution for 1 hour 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 rat brain tissue with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/20,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-2) at 1/20,000 dilution for 1 hour 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 rat hippocampus tissue with Rabbit anti-HMGB1 antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/20,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/20,000 dilution for 1 hour 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 rat hippocampus tissue with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/20,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-2) at 1/20,000 dilution for 1 hour 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.

  • Immunocytochemistry analysis of C2C12 cells labeling HMGB1 with Rabbit anti-HMGB1 antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/500 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-HMGB1 antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.<br /><br />Beta tubulin (<a href="/products/M1305-2" style="font-weight: bold;text-decoration: underline;">M1305-2</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of C2C12 cells labeling HMGB1 with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/500 dilution.

    Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

  • Immunocytochemistry analysis of C6 cells labeling HMGB1 with Rabbit anti-HMGB1 antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/500 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-HMGB1 antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.<br /><br />Beta tubulin (<a href="/products/M1305-2" style="font-weight: bold;text-decoration: underline;">M1305-2</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of C6 cells labeling HMGB1 with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/500 dilution.

    Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

  • Flow cytometric analysis of HCT 116 cells labeling HMGB1.<br /><br />Cells were fixed and permeabilized. Then stained with the primary antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor&trade; 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (<a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

    Flow cytometric analysis of HCT 116 cells labeling HMGB1.

    Cells were fixed and permeabilized. Then stained with the primary antibody (ET1601-2, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

  • Application: IF-Tissue<br /><br />Species: Mouse<br /><br />Site: brain<br /><br />Sample: Paraffin-embedded section<br /><br />Antibody concentration: 1/2,000

    Application: IF-Tissue

    Species: Mouse

    Site: brain

    Sample: Paraffin-embedded section

    Antibody concentration: 1/2,000

  • Application: IHC-Fr<br /><br />Species: Mouse<br /><br />Site: brain<br /><br />Sample: Frozen section<br /><br />Antibody concentration: 1/500<br /><br />Antigen retrieval: Not required

    Application: IHC-Fr

    Species: Mouse

    Site: brain

    Sample: Frozen section

    Antibody concentration: 1/500

    Antigen retrieval: Not required

  • Western blot analysis of HMGB1 on Mouse HMGB1 recombinant protein with Rabbit anti-HMGB1 antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/1,000 dilution.<br /><br />Lysates/proteins at 50 ng/Lane.<br />Exposure time: 59 seconds; ECL: K1801;<br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1601-2" style="font-weight: bold;text-decoration: underline;">ET1601-2</a>) at 1/1,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of HMGB1 on Mouse HMGB1 recombinant protein with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/1,000 dilution.

    Lysates/proteins at 50 ng/Lane.
    Exposure time: 59 seconds; ECL: K1801;
    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1601-2) at 1/1,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Citation

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