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Phospho-RPA32/RPA2 (T21) Recombinant Rabbit Monoclonal Antibody [SN06-36]

Usd: 385

Specification

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Catalog# ET1611-18

Phospho-RPA32/RPA2 (T21) Recombinant Rabbit Monoclonal Antibody [SN06-36]

Application

  • WB

  • IP

  • IF-Cell

Reactivity

  • Human

Predicted reactivity

  • Mouse

  • Rat

Predicted species support after-sales service
Conjugation

  • unconjugated

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

Overview

Product Name

Phospho-RPA32/RPA2 (T21) Recombinant Rabbit Monoclonal Antibody [SN06-36]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic phospho-peptide corresponding to residues surrounding Thr21 of Human RPA32 / RPA2 aa 1-50 / 270.

Species Reactivity

Human (Predicted: Mouse, Rat)

Predicted species support after-sales service

Validated Applications

WB, IP, IF-Cell

Molecular Weight

Predicted band size: 29 kDa

Positive Control

HeLa whole cell lysate, HeLa treated with 1uM Camptothecin for 1 hour whole cell lysate, HEK-293 whole cell lysate, HEK-293 treated with UV for 20 minutes then recover 2 hours whole cell lysate, HEK-293 treated with UV for 40 minutes then recover 2 hours whole cell lysate, HeLa treated with UV for 40 minutes then recover 2 hours whole cell lysate, HeLa, Hela cell lysate, HL-60 cell lysate.

Conjugation

unconjugated

Clone Number

SN06-36

RRID

AB_3069995

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

  • IP

  • Use at an assay dependent concentration.

  • IF-Cell

  • 1:50

Target

Function

As part of the heterotrimeric replication protein A complex (RPA/RP-A), binds and stabilizes single-stranded DNA intermediates that form during DNA replication or upon DNA stress. It prevents their reannealing and in parallel, recruits and activates different proteins and complexes involved in DNA metabolism. Thereby, it plays an essential role both in DNA replication and the cellular response to DNA damage. In the cellular response to DNA damage, the RPA complex controls DNA repair and DNA damage checkpoint activation. Through recruitment of ATRIP activates the ATR kinase a master regulator of the DNA damage response. It is required for the recruitment of the DNA double-strand break repair factors RAD51 and RAD52 to chromatin in response to DNA damage. Also recruits to sites of DNA damage proteins like XPA and XPG that are involved in nucleotide excision repair and is required for this mechanism of DNA repair. Also plays a role in base excision repair (BER) probably through interaction with UNG. Also recruits SMARCAL1/HARP, which is involved in replication fork restart, to sites of DNA damage. May also play a role in telomere maintenance. RPA stimulates 5'-3' helicase activity of BRIP1/FANCJ.

Background References

1. Uchida C et al. p130RB2 positively contributes to ATR activation in response to replication stress via the RPA32-ETAA1 axis. Biochim Biophys Acta Mol Cell Res. 2023 Aug

2. Batenburg NL et al. CSB and SMARCAL1 compete for RPA32 at stalled forks and differentially control the fate of stalled forks in BRCA2-deficient cells. Nucleic Acids Res. 2024 May

Sequence Similarity

Belongs to the replication factor A protein 2 family.

Post-translational Modification

Differentially phosphorylated throughout the cell cycle, becoming phosphorylated at the G1-S transition and dephosphorylated in late mitosis. Mainly phosphorylated at Ser-23 and Ser-29, by cyclin A-CDK2 and cyclin B-CDK1, respectively during DNA replication and mitosis. Dephosphorylation may require the serine/threonine-protein phosphatase 4. Phosphorylation at Ser-23 and Ser-29 is a prerequisite for further phosphorylation. Becomes hyperphosphorylated on additional residues including Ser-4, Ser-8, Thr-21 and Ser-33 in response to DNA damage. Hyperphosphorylation is mediated by ATM, ATR and PRKDC. Primarily recruited to DNA repair nuclear foci as a hypophosphorylated form it undergoes subsequent hyperphosphorylation, catalyzed by ATR. Hyperphosphorylation is required for RAD51 recruitment to chromatin and efficient DNA repair. Phosphorylation at Thr-21 depends upon RFWD3 presence.; DNA damage-induced 'Lys-63'-linked polyubiquitination by PRPF19 mediates ATRIP recruitment to the RPA complex at sites of DNA damage and activation of ATR. Ubiquitinated by RFWD3 at stalled replication forks in response to DNA damage: ubiquitination by RFWD3 does not lead to degradation by the proteasome and promotes removal of the RPA complex from stalled replication forks, promoting homologous recombination.

Subcellular Location

Nucleus, PML body.

UNIPROT

SwissProt: P15927 Human

SwissProt: Q62193 Mouse

SwissProt: Q63528 Rat

Synonyms

60S acidic ribosomal protein P1 antibody

AA409079 antibody

AI325195 antibody

AU020965 antibody

ik:tdsubc_2g1 antibody

M(2)21C antibody

MGC137236 antibody

OTTHUMP00000004008 antibody

p32 antibody

p34 antibody

Expand

Images

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Western blot analysis of Phospho-RPA32/RPA2 (T21) on different lysates with Rabbit anti-Phospho-RPA32/RPA2 (T21) antibody (<a href="/products/ET1611-18" style="font-weight: bold;text-decoration: underline;">ET1611-18</a>) at 1/1,000 dilution.<br /><br />Lane 1: HeLa whole cell lysate<br />Lane 2: HeLa treated with 1uM Camptothecin for 1 hour whole cell lysate<br />Lane 3: HEK-293 whole cell lysate<br />Lane 4: HEK-293 treated with UV for 20 minutes, then recover 2 hours whole cell lysate<br />Lane 5: HEK-293 treated with UV for 40 minutes, then recover 2 hours whole cell lysate<br />Lane 6: HeLa whole cell lysate<br />Lane 7: HeLa treated with UV for 40 minutes, then recover 2 hours whole cell lysate<br /><br />Lysates/proteins at 20 µg/Lane.<br /><br />Predicted band size: 29 kDa<br />Observed band size: 29/35 kDa<br /><br />Exposure time: 1 minute;<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/ET1611-18" style="font-weight: bold;text-decoration: underline;">ET1611-18</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.

    ☑ Cell treatment (CT)

    Western blot analysis of Phospho-RPA32/RPA2 (T21) on different lysates with Rabbit anti-Phospho-RPA32/RPA2 (T21) antibody (ET1611-18) at 1/1,000 dilution.

    Lane 1: HeLa whole cell lysate
    Lane 2: HeLa treated with 1uM Camptothecin for 1 hour whole cell lysate
    Lane 3: HEK-293 whole cell lysate
    Lane 4: HEK-293 treated with UV for 20 minutes, then recover 2 hours whole cell lysate
    Lane 5: HEK-293 treated with UV for 40 minutes, then recover 2 hours whole cell lysate
    Lane 6: HeLa whole cell lysate
    Lane 7: HeLa treated with UV for 40 minutes, then recover 2 hours whole cell lysate

    Lysates/proteins at 20 µg/Lane.

    Predicted band size: 29 kDa
    Observed band size: 29/35 kDa

    Exposure time: 1 minute;

    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 (ET1611-18) 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.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Immunocytochemistry analysis of HeLa cells treated with or without UV for 40 minutes then recover 2 hours labeling Phospho-RPA32/RPA2 (T21) with Rabbit anti-Phospho-RPA32/RPA2 (T21) antibody (<a href="/products/ET1611-18" style="font-weight: bold;text-decoration: underline;">ET1611-18</a>) at 1/50 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-Phospho-RPA32/RPA2 (T21) antibody (<a href="/products/ET1611-18" style="font-weight: bold;text-decoration: underline;">ET1611-18</a>) at 1/50 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 1/100 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.

    ☑ Cell treatment (CT)

    Immunocytochemistry analysis of HeLa cells treated with or without UV for 40 minutes then recover 2 hours labeling Phospho-RPA32/RPA2 (T21) with Rabbit anti-Phospho-RPA32/RPA2 (T21) antibody (ET1611-18) at 1/50 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-Phospho-RPA32/RPA2 (T21) antibody (ET1611-18) at 1/50 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 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

  • Western blot analysis of Phospho-RPA32/RPA2 (T21) on different lysates with Rabbit anti-Phospho-RPA32/RPA2 (T21) antibody (<a href="/products/ET1611-18" style="font-weight: bold;text-decoration: underline;">ET1611-18</a>) at 1/500 dilution.<br /><br />Lane 1: Hela cell lysate<br />Lane 2: HL-60 cell lysate<br /><br />Lysates/proteins at 10 µg/Lane.<br /><br />Predicted band size: 29 kDa<br />Observed band size: 29 kDa (unphosphorylated)<br /><br />Exposure time: 2 minutes;<br /><br />12% 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/ET1611-18" style="font-weight: bold;text-decoration: underline;">ET1611-18</a>) at 1/500 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 1:300,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of Phospho-RPA32/RPA2 (T21) on different lysates with Rabbit anti-Phospho-RPA32/RPA2 (T21) antibody (ET1611-18) at 1/500 dilution.

    Lane 1: Hela cell lysate
    Lane 2: HL-60 cell lysate

    Lysates/proteins at 10 µg/Lane.

    Predicted band size: 29 kDa
    Observed band size: 29 kDa (unphosphorylated)

    Exposure time: 2 minutes;

    12% 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 (ET1611-18) at 1/500 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:300,000 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"

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