Phospho-RPA32/RPA2 (Ser8) Antibody (Rabbit mAb) [B9B12]

Application: Reactivity: Human

Usage Information

Dilution
WB1:1000 IF1:200 - 1:800 FCM1:400 - 1:1600

Application
WB, IF, FCM

Reactivity
Human

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
29 kDa

Datasheet & SDS

Biological Description

Specificity
Phospho-RPA32/RPA2 (Ser8) Antibody (Rabbit mAb) [B9B12] detects endogenous levels of RPA32/RPA2 protein only when phosphorylated at Ser8.

Clone
B9B12

Synonym(s)
REPA2; Replication factor A protein 2; replication protein A2; replication protein A2, 32kDa; RF-A protein 2; RFA2; RP-A p32; RP-A p34; RPA2; RPA32; RPA34

Background
Phospho-RPA32/RPA2 (Ser8) refers to the DNA damage–inducible modification of the 32 kDa subunit of replication protein A within the heterotrimeric RPA complex, a central single-stranded DNA–binding factor required for replication, recombination, checkpoint signaling and all major DNA repair pathways. RPA32 carries multiple N‑terminal serine and threonine sites that form a phospho-code; Ser4 and Ser8, together with Ser33 and other residues, become hyperphosphorylated in response to replication stress and genotoxic insults by phosphatidylinositol 3‑kinase–related kinases including DNA‑PK, ATR and ATM, on a background of prior cell cycle–linked phosphorylation at Ser23 and Ser29 by cyclin-dependent kinases. Phosphorylation of Ser4/Ser8 by DNA‑PK occurs when RPA accumulates on single-stranded DNA at stalled forks and acts as an early replication stress signal that modifies RPA–DNA and RPA–protein interactions; cells lacking DNA‑PK activity or expressing RPA32 mutants in which Ser4/Ser8 cannot be phosphorylated show defective replication checkpoint arrest, premature restart of stalled forks, failure to block late origin firing and increased mitotic catastrophe, indicating that the phospho‑Ser4/Ser8 state is required to enforce ATR–Chk1 checkpoint outputs and protect genome integrity. In this context, replication stress–induced hyperrecombination in Ser4/Ser8 mutants is ATM‑dependent, while other defects are ATM‑independent, placing DNA‑PK–RPA32 Ser4/Ser8 phosphorylation as a key branch point coordinating ATR- and ATM-mediated responses to stalled replication. Phosphorylated RPA32 at Ser4/Ser8 also participates in the regulation of homologous recombination repair: DNA‑PK, ATR and ATM collaboratively modify the RPA32 N‑terminus and p53 in a way that releases p53–RPA complexes, facilitates Rad51 loading and promotes HR repair of double-strand breaks, providing a mechanism for crosstalk between nonhomologous end joining and homologous recombination through coordinated control of p53–RPA interaction. RPA32 Ser4/Ser8 phosphorylation in G2 cells influences TopBP1 and Rad9 accumulation on chromatin, ATM-dependent KAP1 phosphorylation and Rad51 chromatin loading, reinforcing its role as a modulator of checkpoint signaling and recombination factor recruitment beyond the S phase. Phospho-RPA32 Ser8—typically assessed in combination with Ser4—serves as a mechanistic marker of replication stress and DNA damage response, where its status reports on DNA‑PK/ATR/ATM axis activation and functionally tunes replication checkpoint enforcement, fork restart timing, origin usage and the balance between recombination and mitotic catastrophe.

Background

Phospho-RPA32/RPA2 (Ser8) refers to the DNA damage–inducible modification of the 32 kDa subunit of replication protein A within the heterotrimeric RPA complex, a central single-stranded DNA–binding factor required for replication, recombination, checkpoint signaling and all major DNA repair pathways. RPA32 carries multiple N‑terminal serine and threonine sites that form a phospho-code; Ser4 and Ser8, together with Ser33 and other residues, become hyperphosphorylated in response to replication stress and genotoxic insults by phosphatidylinositol 3‑kinase–related kinases including DNA‑PK, ATR and ATM, on a background of prior cell cycle–linked phosphorylation at Ser23 and Ser29 by cyclin-dependent kinases. Phosphorylation of Ser4/Ser8 by DNA‑PK occurs when RPA accumulates on single-stranded DNA at stalled forks and acts as an early replication stress signal that modifies RPA–DNA and RPA–protein interactions; cells lacking DNA‑PK activity or expressing RPA32 mutants in which Ser4/Ser8 cannot be phosphorylated show defective replication checkpoint arrest, premature restart of stalled forks, failure to block late origin firing and increased mitotic catastrophe, indicating that the phospho‑Ser4/Ser8 state is required to enforce ATR–Chk1 checkpoint outputs and protect genome integrity. In this context, replication stress–induced hyperrecombination in Ser4/Ser8 mutants is ATM‑dependent, while other defects are ATM‑independent, placing DNA‑PK–RPA32 Ser4/Ser8 phosphorylation as a key branch point coordinating ATR- and ATM-mediated responses to stalled replication. Phosphorylated RPA32 at Ser4/Ser8 also participates in the regulation of homologous recombination repair: DNA‑PK, ATR and ATM collaboratively modify the RPA32 N‑terminus and p53 in a way that releases p53–RPA complexes, facilitates Rad51 loading and promotes HR repair of double-strand breaks, providing a mechanism for crosstalk between nonhomologous end joining and homologous recombination through coordinated control of p53–RPA interaction. RPA32 Ser4/Ser8 phosphorylation in G2 cells influences TopBP1 and Rad9 accumulation on chromatin, ATM-dependent KAP1 phosphorylation and Rad51 chromatin loading, reinforcing its role as a modulator of checkpoint signaling and recombination factor recruitment beyond the S phase. Phospho-RPA32 Ser8—typically assessed in combination with Ser4—serves as a mechanistic marker of replication stress and DNA damage response, where its status reports on DNA‑PK/ATR/ATM axis activation and functionally tunes replication checkpoint enforcement, fork restart timing, origin usage and the balance between recombination and mitotic catastrophe.

References
https://pubmed.ncbi.nlm.nih.gov/22797063/ https://pubmed.ncbi.nlm.nih.gov/24819595/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%