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CENPB Mouse Monoclonal Antibody [A5F5]

Usd: 350

Specification

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Catalog# HA600070

CENPB Mouse Monoclonal Antibody [A5F5]

Application

  • WB

  • IF-Cell

  • IHC-P

  • IP

Reactivity

  • Human

  • Mouse

  • Rat

Conjugation

  • unconjugated

Overview

Product Name

CENPB Mouse Monoclonal Antibody [A5F5]

Antibody Type

Mouse Monoclonal Antibody

Immunogen

Recombinant protein within human CENPB aa 1-200 / 599.

Species Reactivity

Human, Mouse, Rat

Validated Applications

WB, IF-Cell, IHC-P, IP

Target Molecular Weight

Predicted band size: 65 kDa

Positive Control

A431 (Human epidermoid carcinoma skin squamous cell) cell lysate, Daudi (Human Burkitt's lymphoma cell) cell lysate, NIH/3T3 (Mouse fibroblast) cell lysate, PC-12 (Rat pheochromocytoma cell (undifferentiated)) cell lysate, SK-OV-3, human kidney tissue, human skin tissue.

Conjugation

unconjugated

Clone Number

A5F5

RRID

AB_3071687

Product Features

Form

Liquid

Concentration

2 mg/mL.(The concentration of this product may be batch-dependent)

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), 0.2% BSA, 50% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgG2a

Purification Method

Protein G affinity purified.

Application Dilution

  • WB

  • 1:5,000

  • IF-Cell

  • 1:400

  • IHC-P

  • 1:100-1:500

  • IP

  • Use at an assay dependent concentration.

Target

Function

This gene product is a highly conserved protein that facilitates centromere formation. It is a DNA-binding protein that is derived from transposases of the pogo DNA transposon family. It contains a helix-loop-helix DNA binding motif at the N-terminus, and a dimerization domain at the C-terminus. The DNA binding domain recognizes and binds a 17-bp sequence (CENP-B box) in the centromeric alpha satellite DNA. This protein is proposed to play an important role in the assembly of specific centromere structures in interphase nuclei and on mitotic chromosomes. It is also considered a major centromere autoantigen recognized by sera from patients with anti-centromere antibodies.

Background References

1. Otake K. et. al. CENP-B creates alternative epigenetic chromatin states permissive for CENP-A or heterochromatin assembly. J Cell Sci. 2020 Aug

2. Ohzeki JI. et. al. Human artificial chromosome: Chromatin assembly mechanisms and CENP-B. Exp Cell Res. 2020 Apr

Subcellular Location

Nucleus, centromere.

UNIPROT

SwissProt: P07199 Human

Synonyms

CENP B antibody

CENP-B antibody

CENPB antibody

CENPB_HUMAN antibody

centromere autoantigen B antibody

Centromere protein B antibody

Centromere protein B, 80kDa antibody

Major centromere autoantigen B antibody

Images

  • Western blot analysis of CENPB on different lysates with Mouse anti-CENPB antibody (<a href="/products/HA600070" style="font-weight: bold;text-decoration: underline;">HA600070</a>) at 1/5,000 dilution.<br /><br />Lane 1: A431 (Human epidermoid carcinoma skin squamous cell) cell lysate<br />Lane 2: Daudi (Human Burkitt's lymphoma cell) cell lysate<br />Lane 3: NIH/3T3 (Mouse fibroblast) cell lysate<br />Lane 4: PC-12 (Rat pheochromocytoma cell (undifferentiated)) cell lysate<br /><br />Lysates/proteins at 15 µg/Lane.<br />Exposure time: 1 minute 50 seconds; ECL: K1801<br /><br />Blocking: 5% NFDM/TBST, 1 hour at room temperature<br />Primary antibody: <a href="/products/HA600070" style="font-weight: bold;text-decoration: underline;">HA600070</a>, 1/5,000 in primary antibody dilution buffer (<a href="/products/K1803" style="font-weight: bold;text-decoration: underline;">K1803</a>), overnight at 4 ℃<br />Secondary antibody: Goat anti-Mouse IgG-HRP (<a href="/products/HA1006" style="font-weight: bold;text-decoration: underline;">HA1006</a>), 1/50,000 in 5% NFDM/TBST, 1 hour at room temperature<br /><br />Predicted band size: 65 kDa<br />Observed band size: 77 kDa

    Western blot analysis of CENPB on different lysates with Mouse anti-CENPB antibody (HA600070) at 1/5,000 dilution.

    Lane 1: A431 (Human epidermoid carcinoma skin squamous cell) cell lysate
    Lane 2: Daudi (Human Burkitt's lymphoma cell) cell lysate
    Lane 3: NIH/3T3 (Mouse fibroblast) cell lysate
    Lane 4: PC-12 (Rat pheochromocytoma cell (undifferentiated)) cell lysate

    Lysates/proteins at 15 µg/Lane.
    Exposure time: 1 minute 50 seconds; ECL: K1801

    Blocking: 5% NFDM/TBST, 1 hour at room temperature
    Primary antibody: HA600070, 1/5,000 in primary antibody dilution buffer (K1803), overnight at 4 ℃
    Secondary antibody: Goat anti-Mouse IgG-HRP (HA1006), 1/50,000 in 5% NFDM/TBST, 1 hour at room temperature

    Predicted band size: 65 kDa
    Observed band size: 77 kDa

  • Immunocytochemistry analysis of SK-OV-3 cells labeling CENPB with Mouse anti-CENPB antibody (<a href="/products/HA600070" style="font-weight: bold;text-decoration: underline;">HA600070</a>) at 1/400 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 Mouse anti-CENPB antibody (<a href="/products/HA600070" style="font-weight: bold;text-decoration: underline;">HA600070</a>) at 1/400 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 488, <a href="/products/HA1125" style="font-weight: bold;text-decoration: underline;">HA1125</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/ET1602-4" style="font-weight: bold;text-decoration: underline;">ET1602-4</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 594, <a href="/products/HA1122" style="font-weight: bold;text-decoration: underline;">HA1122</a>) were used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of SK-OV-3 cells labeling CENPB with Mouse anti-CENPB antibody (HA600070) at 1/400 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 Mouse anti-CENPB antibody (HA600070) at 1/400 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 488, HA1125) 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 (ET1602-4, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) were used as the secondary antibody at 1/1,000 dilution.

  • Immunohistochemical analysis of paraffin-embedded human kidney tissue using anti-CENPB antibody. 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 30 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/HA600070" style="font-weight: bold;text-decoration: underline;">HA600070</a>, 1/400) 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-CENPB antibody. 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 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA600070, 1/400) 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 skin tissue using anti-CENPB antibody. 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 30 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/HA600070" style="font-weight: bold;text-decoration: underline;">HA600070</a>, 1/100) 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 skin tissue using anti-CENPB antibody. 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 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA600070, 1/100) 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.

  • CENPB was immunoprecipitated from 0.4 mg Hela whole cell lysates with <a href="/products/HA600070" style="font-weight: bold;text-decoration: underline;">HA600070</a> at 2 μg/mL. Western blot was performed from the immunoprecipitate using <a href="/products/HA600070" style="font-weight: bold;text-decoration: underline;">HA600070</a> at 1/500 dilution for 45 minutes at room temperature. Goat anti-Mouse IgG - HRP Secondary Antibody (<a href="/products/HA1006" style="font-weight: bold;text-decoration: underline;">HA1006</a>) was used at 1:100,000 dilution for 30 minutes at room temperature.<br /><br />Lane 1: Hela whole cell lysates at 4 μg;<br />Lane 2: CENPB (<a href="/products/HA600070" style="font-weight: bold;text-decoration: underline;">HA600070</a>) IP in Hela whole cell lysates;<br />Lane 3: Mouse IgG instead of CENPB (<a href="/products/HA600070" style="font-weight: bold;text-decoration: underline;">HA600070</a>) in Hela whole cell lysates.<br /><br />Predicted band size: 65 kDa<br />Observed band size: 85 kDa<br /><br />Exposure time: 2 minutes;<br /><br />8% SDS-PAGE gel.

    CENPB was immunoprecipitated from 0.4 mg Hela whole cell lysates with HA600070 at 2 μg/mL. Western blot was performed from the immunoprecipitate using HA600070 at 1/500 dilution for 45 minutes at room temperature. Goat anti-Mouse IgG - HRP Secondary Antibody (HA1006) was used at 1:100,000 dilution for 30 minutes at room temperature.

    Lane 1: Hela whole cell lysates at 4 μg;
    Lane 2: CENPB (HA600070) IP in Hela whole cell lysates;
    Lane 3: Mouse IgG instead of CENPB (HA600070) in Hela whole cell lysates.

    Predicted band size: 65 kDa
    Observed band size: 85 kDa

    Exposure time: 2 minutes;

    8% SDS-PAGE gel.

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