Phospho-RPA32/RPA2 (Ser4/8) Antibody [M17K6]

Application: Reactivity: Human

Usage Information

Dilution
WB1:1000 IP1:1000 IF1:5000 FCM1:2000

Application
WB, IP, 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 (Ser4/8) Antibody [M17K6] detects endogenous levels of total RPA32 protein only when it is phosphorylated at Ser4/8.

Clone
M17K6

Synonym(s)
Replication protein A 32 kDa subunit, RP-A p32, Replication factor A protein 2 (RF-A protein 2), Replication protein A 34 kDa subunit (RP-A p34) RPA2, REPA2, RPA32, RPA34

Background
Phospho‑RPA32 (Ser4/8) marks a hyperphosphorylated, damage‑responsive state of the 32‑kDa subunit of replication protein A in which DNA‑PK–dependent modification of two N‑terminal serines reprograms RPA’s functions at stalled replication forks and double‑strand breaks, coordinating checkpoint signaling, recombination, fork restart, and the decision between recovery and mitotic catastrophe. RPA32 resides in the trimeric RPA complex together with RPA70 and RPA14 and contributes an N‑terminal regulatory tail that is phosphorylated by PIKK kinases and CDKs and a central OB‑fold ssDNA‑binding domain; Ser4 and Ser8 lie in this unstructured N‑terminus and are among the earliest sites to become phosphorylated when replication stress generates extended RPA‑coated ssDNA. Under replication stress, ATR activation by RPA–ssDNA complexes triggers a first wave of RPA32 phosphorylation at sites such as Ser33 that supports Chk1 activation and replication arrest, while DNA‑PK subsequently phosphorylates Ser4/Ser8, and cells either lacking DNA‑PK activity or expressing a Ser4/8A mutant show nearly identical phenotypes, including defective replication checkpoint arrest, premature fork restart, failure to suppress late origin firing, ATM‑dependent hyper‑recombination, and increased mitotic catastrophe, indicating that Ser4/8 phosphorylation is a critical effector branch of DNA‑PK signaling at stalled forks. These modifications influence how RPA interfaces with other repair factors: Ser4/8 phosphorylation promotes proper MRE11 and TopBP1 phosphorylation and helps maintain ATR–Chk1 signaling, while also modulating recruitment and turnover of recombination proteins such as RAD51, so that loss of this modification leads to excessive homologous recombination events that compromise genome stability rather than resolving damage faithfully.

Background

Phospho‑RPA32 (Ser4/8) marks a hyperphosphorylated, damage‑responsive state of the 32‑kDa subunit of replication protein A in which DNA‑PK–dependent modification of two N‑terminal serines reprograms RPA’s functions at stalled replication forks and double‑strand breaks, coordinating checkpoint signaling, recombination, fork restart, and the decision between recovery and mitotic catastrophe. RPA32 resides in the trimeric RPA complex together with RPA70 and RPA14 and contributes an N‑terminal regulatory tail that is phosphorylated by PIKK kinases and CDKs and a central OB‑fold ssDNA‑binding domain; Ser4 and Ser8 lie in this unstructured N‑terminus and are among the earliest sites to become phosphorylated when replication stress generates extended RPA‑coated ssDNA. Under replication stress, ATR activation by RPA–ssDNA complexes triggers a first wave of RPA32 phosphorylation at sites such as Ser33 that supports Chk1 activation and replication arrest, while DNA‑PK subsequently phosphorylates Ser4/Ser8, and cells either lacking DNA‑PK activity or expressing a Ser4/8A mutant show nearly identical phenotypes, including defective replication checkpoint arrest, premature fork restart, failure to suppress late origin firing, ATM‑dependent hyper‑recombination, and increased mitotic catastrophe, indicating that Ser4/8 phosphorylation is a critical effector branch of DNA‑PK signaling at stalled forks. These modifications influence how RPA interfaces with other repair factors: Ser4/8 phosphorylation promotes proper MRE11 and TopBP1 phosphorylation and helps maintain ATR–Chk1 signaling, while also modulating recruitment and turnover of recombination proteins such as RAD51, so that loss of this modification leads to excessive homologous recombination events that compromise genome stability rather than resolving damage faithfully.

References
https://pubmed.ncbi.nlm.nih.gov/24819595/ https://pubmed.ncbi.nlm.nih.gov/22977173/

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%