DNA-PKcs Antibody (Rabbit mAb) [H12B18]

Application: Reactivity: Human

Usage Information

Dilution
WB1:1000 IHC1:400 - 1:1600 IF1:50 - 1:100 FCM1:100 - 1:400

Application
WB, IHC, 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
469 kDa

Datasheet & SDS

Biological Description

Specificity
DNA-PKcs Antibody (Rabbit mAb) [H12B18] detects endogenous levels of total DNA-PKcs protein.

Clone
H12B18

Synonym(s)
DNA-PK; DNA-PK catalytic subunit; DNA-PKC; DNA-PKcs; DNAPK; DNAPKc; DNPK1; HYRC; HYRC1; IMD26; p350; p460; PRKDC; protein kinase, DNA-activated, catalytic subunit; XRCC7

Background
DNA‑PKcs (DNA‑dependent protein kinase catalytic subunit, PRKDC) is the largest serine/threonine kinase of the phosphatidylinositol 3‑kinase–related kinase family and forms, together with the Ku70/Ku80 heterodimer, the DNA‑PK holoenzyme that initiates and orchestrates nonhomologous end‑joining repair of DNA double‑strand breaks by detecting paired broken ends, assembling a synaptic repair complex and directing a cascade of phosphorylation events on itself and multiple DNA repair factors. The protein is composed of extensive HEAT‑repeat arms that curve around DNA and a globular head containing the kinase, FAT and FATC domains, and structural analyses of NHEJ synaptic complexes show that Ku70/80 first encircles one DNA end and recruits DNA‑PKcs to form a long‑range synapsis, with a conserved YRPD motif and flexible HEAT repeats in DNA‑PKcs pivoting to bring two ends into close proximity and activate the kinase when both ends are engaged. Activated DNA‑PKcs phosphorylates a broad panel of substrates, including Ku70/80, XRCC4, XLF, Artemis, H2AX, p53 and other DDR components, thereby promoting end tethering, processing of hairpins or incompatible ends, recruitment of ligase IV and coordination with ATM‑dependent signaling, while its own autophosphorylation at two major clusters, centered on Thr2609 and Ser2056, dynamically regulates complex assembly, synapsis stability and end release. Mutational and phospho‑cluster analyses indicate that the Thr2609 cluster is critical for proper end processing and pathway choice: phosphorylation at this cluster promotes resection, suppresses aberrant end ligation and is required for efficient double‑strand break repair, with DNA‑PKcs‑deficient or Thr2609‑mutant cells showing impaired NHEJ and altered use of backup repair mechanisms. The Ser2056 cluster is primarily involved in regulating synaptic complex disassembly and end‑ligase access, and coordinated phosphorylation of Ser2056 and Thr2609 provides a temporal control mechanism in which DNA‑PKcs initially phosphorylates external substrates to stabilize and configure the repair machinery and subsequently autophosphorylates to reduce its own end‑binding affinity and permit completion of ligation and release of DNA ends. DNA‑PKcs and ATM have complementary functions in double‑strand break repair: both kinases are activated by breaks and share targets, but ATM deficiency leads to timely yet incomplete repair with higher illegitimate recombination, whereas DNA‑PKcs loss causes slower repair and accumulation of simple and complex chromosomal rearrangements; simultaneous deficiency in V(D)J and class‑switch recombination markedly increases translocations and deletions, showing that fast rejoining by DNA‑PKcs and end‑fidelity assurance by ATM are jointly required for genome integrity. DNA‑PKcs is also essential for physiological recombination in lymphocytes, and its absence produces severe combined immunodeficiency due to failure of V(D)J joining, underlining its central role in immune system development. Beyond canonical NHEJ, recent work has revealed non‑canonical DNA‑PKcs functions involving transcriptional regulation, telomere maintenance, metabolic control and immune signaling, and reviews highlight that DNA‑PKcs overexpression or hyperactivation in multiple solid and hematologic malignancies contributes to radioresistance, chemoresistance, proliferative signaling and protumorigenic inflammation, positioning DNA‑PKcs as a targetable oncoprotein and driving development of selective DNA‑PKcs inhibitors for combination cancer therapy.

Background

DNA‑PKcs (DNA‑dependent protein kinase catalytic subunit, PRKDC) is the largest serine/threonine kinase of the phosphatidylinositol 3‑kinase–related kinase family and forms, together with the Ku70/Ku80 heterodimer, the DNA‑PK holoenzyme that initiates and orchestrates nonhomologous end‑joining repair of DNA double‑strand breaks by detecting paired broken ends, assembling a synaptic repair complex and directing a cascade of phosphorylation events on itself and multiple DNA repair factors. The protein is composed of extensive HEAT‑repeat arms that curve around DNA and a globular head containing the kinase, FAT and FATC domains, and structural analyses of NHEJ synaptic complexes show that Ku70/80 first encircles one DNA end and recruits DNA‑PKcs to form a long‑range synapsis, with a conserved YRPD motif and flexible HEAT repeats in DNA‑PKcs pivoting to bring two ends into close proximity and activate the kinase when both ends are engaged. Activated DNA‑PKcs phosphorylates a broad panel of substrates, including Ku70/80, XRCC4, XLF, Artemis, H2AX, p53 and other DDR components, thereby promoting end tethering, processing of hairpins or incompatible ends, recruitment of ligase IV and coordination with ATM‑dependent signaling, while its own autophosphorylation at two major clusters, centered on Thr2609 and Ser2056, dynamically regulates complex assembly, synapsis stability and end release. Mutational and phospho‑cluster analyses indicate that the Thr2609 cluster is critical for proper end processing and pathway choice: phosphorylation at this cluster promotes resection, suppresses aberrant end ligation and is required for efficient double‑strand break repair, with DNA‑PKcs‑deficient or Thr2609‑mutant cells showing impaired NHEJ and altered use of backup repair mechanisms. The Ser2056 cluster is primarily involved in regulating synaptic complex disassembly and end‑ligase access, and coordinated phosphorylation of Ser2056 and Thr2609 provides a temporal control mechanism in which DNA‑PKcs initially phosphorylates external substrates to stabilize and configure the repair machinery and subsequently autophosphorylates to reduce its own end‑binding affinity and permit completion of ligation and release of DNA ends. DNA‑PKcs and ATM have complementary functions in double‑strand break repair: both kinases are activated by breaks and share targets, but ATM deficiency leads to timely yet incomplete repair with higher illegitimate recombination, whereas DNA‑PKcs loss causes slower repair and accumulation of simple and complex chromosomal rearrangements; simultaneous deficiency in V(D)J and class‑switch recombination markedly increases translocations and deletions, showing that fast rejoining by DNA‑PKcs and end‑fidelity assurance by ATM are jointly required for genome integrity. DNA‑PKcs is also essential for physiological recombination in lymphocytes, and its absence produces severe combined immunodeficiency due to failure of V(D)J joining, underlining its central role in immune system development. Beyond canonical NHEJ, recent work has revealed non‑canonical DNA‑PKcs functions involving transcriptional regulation, telomere maintenance, metabolic control and immune signaling, and reviews highlight that DNA‑PKcs overexpression or hyperactivation in multiple solid and hematologic malignancies contributes to radioresistance, chemoresistance, proliferative signaling and protumorigenic inflammation, positioning DNA‑PKcs as a targetable oncoprotein and driving development of selective DNA‑PKcs inhibitors for combination cancer therapy.

References
https://pubmed.ncbi.nlm.nih.gov/28118114/ https://pubmed.ncbi.nlm.nih.gov/25550082/

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%