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dUTPase Recombinant Rabbit Monoclonal Antibody [JE66-11]

Usd: 385

Specification

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Catalog# HA721389

dUTPase Recombinant Rabbit Monoclonal Antibody [JE66-11]

Application

  • WB

  • IHC-P

  • IF-Cell

  • FC

Reactivity

  • Human

Conjugation

  • unconjugated

Overview

Product Name

dUTPase Recombinant Rabbit Monoclonal Antibody [JE66-11]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Recombinant protein within Human dUTPase aa 153-252 / 252.

Species Reactivity

Human

Validated Applications

WB, IHC-P, IF-Cell, FC

Molecular Weight

Predicted band size: 27 kDa

Positive Control

HeLa cell lysate, A549 cell lysate, Jurkat cell lysate, LO2 cell lysate, HepG2 cell lysate, K-562 cell lysate, HEK-293 cell lysate, A431 cell lysate, human liver tissue lysate, human esophagus tissue, human spleen tissue, HCT116.

Conjugation

unconjugated

Clone Number

JE66-11

RRID

AB_3072506

Product Features

Form

Liquid

Concentration

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). 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

  • 1:1,000-1:2,000

  • IHC-P

  • 1:1,000

  • IF-Cell

  • 1:100

  • FC

  • 1:500-1:1,000

Target

Function

DUT is a member of the dUTPase family, which is known for catalyzing the pyrophosphoralysis of dUTP into dUMP and inorganic pyrophosphate. This function contributes to DNA replication and repair via de novo thymidylate biosynthesis, as the dUMP product is methylated by thymidylate synthase (TS) to form dTMP, which is then phosphorylated to dTTP. DUT is also crucial for maintaining genome integrity by reducing cellular dUTP levels, thereby preventing the repeated cycles of uracil misincorporation into DNA and DNA repair-mediated strand breaks that would lead to cell death. In addition to their different localizations, the two DUT isoforms display different expression patterns: while DUT-M is constitutively expressed, DUT-N is under cell cycle control and notably upregulated during S phase. These expression patterns correspond with their roles in the DNA replication cycle of their respective genomes, and thus indicate different regulatory mechanisms affecting each isoform.

Background References

1. Otsuki S et al. Monocyte-released HERV-K dUTPase engages TLR4 and MCAM causing endothelial mesenchymal transition. JCI Insight. 2021 Aug

2. Williams MV et al. EBV dUTPase: A Novel Modulator of Inflammation and the Tumor Microenvironment in EBV-Associated Malignancies. Cancers (Basel). 2023 Jan

Subcellular Location

Nucleus; Mitochondrion.

UNIPROT

SwissProt: P33316 Human

Synonyms

Deoxyuridine 5' triphosphate nucleotidohydrolase, nuclear isoform antibody

Deoxyuridine 5'-triphosphate nucleotidohydrolase, mitochondrial antibody

Deoxyuridine triphosphatase antibody

dut antibody

DUT_HUMAN antibody

dUTP nucleotidohydrolase antibody

dUTP pyrophosphatase antibody

dUTPase antibody

FLJ20622 antibody

Images

  • Western blot analysis of dUTPase on different lysates with Rabbit anti-dUTPase antibody (<a href="/products/HA721389" style="font-weight: bold;text-decoration: underline;">HA721389</a>) at 1/1,000 dilution.<br /><br />Lane 1: HeLa cell lysate (15 µg/Lane)<br />Lane 2: A549 cell lysate (15 µg/Lane)<br />Lane 3: Jurkat cell lysate (15 µg/Lane)<br />Lane 4: LO2 cell lysate (15 µg/Lane)<br />Lane 5: HepG2 cell lysate (15 µg/Lane)<br />Lane 6: K-562 cell lysate (15 µg/Lane)<br />Lane 7: HEK-293 cell lysate (15 µg/Lane)<br />Lane 8: A431 cell lysate (15 µg/Lane)<br />Lane 9: Human liver tissue lysate (30 µg/Lane)<br /><br />Predicted band size: 27 kDa<br />Observed band size: 20 kDa<br /><br />Exposure time: 3 minutes;<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/HA721389" style="font-weight: bold;text-decoration: underline;">HA721389</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1:100,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of dUTPase on different lysates with Rabbit anti-dUTPase antibody (HA721389) at 1/1,000 dilution.

    Lane 1: HeLa cell lysate (15 µg/Lane)
    Lane 2: A549 cell lysate (15 µg/Lane)
    Lane 3: Jurkat cell lysate (15 µg/Lane)
    Lane 4: LO2 cell lysate (15 µg/Lane)
    Lane 5: HepG2 cell lysate (15 µg/Lane)
    Lane 6: K-562 cell lysate (15 µg/Lane)
    Lane 7: HEK-293 cell lysate (15 µg/Lane)
    Lane 8: A431 cell lysate (15 µg/Lane)
    Lane 9: Human liver tissue lysate (30 µg/Lane)

    Predicted band size: 27 kDa
    Observed band size: 20 kDa

    Exposure time: 3 minutes;

    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 (HA721389) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.

  • Immunohistochemical analysis of paraffin-embedded human esophagus tissue with Rabbit anti-dUTPase antibody (<a href="/products/HA721389" style="font-weight: bold;text-decoration: underline;">HA721389</a>) at 1/1,000 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/HA721389" style="font-weight: bold;text-decoration: underline;">HA721389</a>) at 1/1,000 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 esophagus tissue with Rabbit anti-dUTPase antibody (HA721389) at 1/1,000 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 (HA721389) at 1/1,000 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 spleen tissue with Rabbit anti-dUTPase antibody (<a href="/products/HA721389" style="font-weight: bold;text-decoration: underline;">HA721389</a>) at 1/1,000 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/HA721389" style="font-weight: bold;text-decoration: underline;">HA721389</a>) at 1/1,000 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 spleen tissue with Rabbit anti-dUTPase antibody (HA721389) at 1/1,000 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 (HA721389) at 1/1,000 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 HCT116 cells labeling dUTPase with Rabbit anti-dUTPase antibody (<a href="/products/HA721389" style="font-weight: bold;text-decoration: underline;">HA721389</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-dUTPase antibody (<a href="/products/HA721389" style="font-weight: bold;text-decoration: underline;">HA721389</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. 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 1/200 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 HCT116 cells labeling dUTPase with Rabbit anti-dUTPase antibody (HA721389) 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-dUTPase antibody (HA721389) 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. Nuclear DNA was labelled in blue with DAPI.

    Beta tubulin (M1305-2, red) was stained at 1/200 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 HCT116 cells labeling dUTPase.<br /><br />Cells were fixed and permeabilized. Then stained with the primary antibody (<a href="/products/HA721389" style="font-weight: bold;text-decoration: underline;">HA721389</a>, 1ug/ml) (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 HCT116 cells labeling dUTPase.

    Cells were fixed and permeabilized. Then stained with the primary antibody (HA721389, 1ug/ml) (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).

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