dnapkcs Antibody

What is DNA-PKcs and why are antibodies against it important in research?

DNA-PKcs is a large 460-469 kDa nuclear protein encoded by the PRKDC gene that acts as a molecular sensor for DNA damage. It belongs to the PI3K kinase family and plays vital roles in DNA double-strand break repair via non-homologous end joining (NHEJ), V(D)J recombination, and immune system development .

Antibodies against DNA-PKcs are crucial research tools because they enable:

• Detection of DNA-PKcs expression patterns across different cell types and tissues

• Monitoring DNA-PKcs localization and recruitment to DNA damage sites

• Studying activation through detection of specific phosphorylation events

• Investigating DNA-PKcs interactions with other proteins in repair complexes

• Assessing the efficacy of DNA-PKcs inhibitors in cancer therapeutic research

DNA-PKcs dysregulation has been implicated in multiple solid and hematologic tumors, including chronic lymphomas, colon, prostate, breast, cervical, and brain cancers, with overexpression often correlating with poor prognosis . This makes DNA-PKcs antibodies valuable tools for both basic research and translational applications.

What experimental applications are DNA-PKcs antibodies most commonly used for?

DNA-PKcs antibodies are utilized across multiple experimental platforms:

Beyond these common applications, specialized uses include:

• Chromatin immunoprecipitation (ChIP) to study DNA-PKcs recruitment to chromatin

• Co-immunoprecipitation to identify protein interaction partners

• Phospho-specific detection to monitor activation status

• High-throughput screening for DNA-PKcs inhibitor development

Each application requires optimization for the specific experimental system being used, with particular attention to antibody specificity and validation.

What are the critical phosphorylation sites of DNA-PKcs and how can they be detected?

DNA-PKcs undergoes autophosphorylation at specific sites that regulate its function in DNA repair. The most crucial sites identified include:

These phosphorylation events serve as indicators of DNA-PKcs activation following DNA damage. Phospho-specific antibodies that recognize these sites provide powerful tools for monitoring DNA-PKcs activity in response to DNA-damaging agents or inhibitor treatments .

For optimal detection of these phosphorylation sites:

• Induce DNA damage with ionizing radiation or genotoxic agents

• Collect samples at appropriate time points (typically 15-60 minutes post-damage)

• Use phospho-specific antibodies that do not bind unphosphorylated DNA-PKcs

• Include appropriate controls (phosphatase treatment, kinase inhibitors)

These phospho-specific antibodies have enabled researchers to track the temporal dynamics of DNA-PKcs activation in various experimental systems .

How can DNA-PKcs antibodies be optimized for detection in different cellular fractions?

The large size and compartment-specific functions of DNA-PKcs require specialized approaches for detection in different cellular fractions:

For nuclear fraction analysis:

• Use gentle lysis buffers (e.g., HEPES-based with 0.1% NP-40) to preserve nuclear integrity

• Employ nuclear extraction protocols with DNase treatment to release chromatin-bound DNA-PKcs

• Use phosphatase inhibitors to preserve phosphorylation status

• Consider sonication to release tightly bound DNA-PKcs from chromatin fragments

For cytoplasmic fraction analysis:

• Use hypotonic buffers without detergents for initial fractionation

• Verify fraction purity with markers (e.g., GAPDH for cytoplasm, lamin for nuclear fraction)

• Consider that non-canonical functions of DNA-PKcs may involve cytoplasmic localization, particularly in immune responses

For chromatin-bound fraction:

• Employ sequential extraction with increasing salt concentrations

• Consider micrococcal nuclease treatment to release DNA-bound proteins

• Use specialized extraction buffers containing 300-420 mM NaCl

Optimization strategies should include validation with appropriate subcellular markers and comparison with total cellular extracts to ensure complete recovery .

What approaches can be used to study DNA-PKcs in the context of viral DNA sensing?

Recent research has revealed DNA-PKcs as an essential component of the cGAS/STING-dependent interferon response to viral DNA. When designing experiments to study this non-canonical function:

• Experimental design considerations:

  • Use DNA viruses (HSV-1, HTLV-1, HBV, KSHV) as mentioned in search result

  • Compare responses in DNA-PKcs-proficient versus deficient cells

  • Monitor interferon responses using reporter systems or ELISA

  • Track DNA-PKcs recruitment to viral DNA using immunofluorescence

• Co-localization studies:

  • Use DNA-PKcs antibodies alongside markers for cytosolic DNA sensors (cGAS, STING)

  • Perform dual immunofluorescence to detect co-localization with viral components

  • Employ super-resolution microscopy for detailed spatial analysis

  • Use proximity ligation assays to confirm protein-protein interactions

• Biochemical approaches:

  • Immunoprecipitate DNA-PKcs to identify viral DNA or proteins in the complex

  • Examine phosphorylation status using phospho-specific antibodies

  • Use chromatin immunoprecipitation to identify binding to viral DNA genomes

• Functional validation:

  • Compare interferon responses in cells treated with DNA-PKcs inhibitors

  • Use CRISPR/Cas9 to generate DNA-PKcs knockout cells for comparison

  • Assess viral replication efficiency under different conditions

These approaches have revealed that DNA-PKcs functions beyond its canonical role in DNA repair, participating in innate immune responses against both cytoplasmic and nuclear-replicating DNA viruses .

How can phospho-specific DNA-PKcs antibodies be utilized to evaluate inhibitor efficacy?

DNA-PKcs inhibitors are being developed as potential cancer therapeutics, particularly as adjuncts to radiotherapy and chemotherapy. Phospho-specific antibodies provide crucial tools for evaluating inhibitor efficacy:

• Experimental design for inhibitor assessment:

  • Establish baseline phosphorylation levels using phospho-specific antibodies

  • Treat cells with inhibitor compounds (e.g., DA-143, NU7441) at varying concentrations

  • Induce DNA damage using radiation or DNA-damaging agents

  • Measure changes in phosphorylation at Thr2609 and Ser2056 sites

• Quantitative evaluation methods:

  • Western blot with phospho-specific antibodies followed by densitometry

  • High-throughput ELISA-based methods for multiple sample analysis

  • Flow cytometry for single-cell resolution of phosphorylation status

  • Antibody microarrays for multiplexed phosphorylation analysis

• Functional correlation assessment:

  • Associate inhibition of phosphorylation with functional outcomes:

    • DNA repair capacity using comet assays or γH2AX foci resolution

    • Cell survival following DNA damage

    • Effects on T cell function for immunological applications

• Comparative inhibitor analysis:

  • Compare novel inhibitors (e.g., DA-143) with established compounds

  • Determine IC50 values based on phosphorylation inhibition

  • Assess bioavailability parameters alongside phosphorylation inhibition

The novel DNA-PKcs inhibitor DA-143 demonstrates improved aqueous solubility compared to previous inhibitors while achieving an IC50 of 2.5 nM, making it promising for both preclinical and clinical development .

How can DNA-PKcs antibodies be employed to investigate its role in T cell function?

Beyond its well-established role in DNA repair, DNA-PKcs has emerged as a critical regulator of T cell function. Researchers can investigate this role using the following approaches:

• Expression and activation analysis:

  • Use Western blot and flow cytometry to assess DNA-PKcs expression in resting versus activated T cells

  • Employ phospho-specific antibodies to track activation status during T cell stimulation

  • Compare expression patterns across T cell subsets (naive, memory, effector)

• Functional studies with inhibitors:

  • Treat T cells with DNA-PKcs inhibitors (e.g., NU7441) before activation

  • Measure activation markers (CD69, CD25), proliferation, and cytokine production

  • Use phospho-specific antibodies to confirm inhibition of DNA-PKcs activity

• Mechanistic investigations:

  • Perform immunoprecipitation with DNA-PKcs antibodies to identify T cell-specific interaction partners

  • Use ChIP-seq to identify DNA-PKcs binding sites in T cell-specific genes

  • Study the relationship between DNA-PKcs and transcription factors like Egr1

• In vivo implications:

  • Use immunohistochemistry to examine DNA-PKcs expression in lymphoid tissues

  • Correlate DNA-PKcs activity with T cell responses in disease models

  • Assess potential for DNA-PKcs inhibitors in immunological disorders

Research has shown that DNA-PKcs inhibition impedes proper T cell response post-stimulation and prevents T cells from producing inflammatory cytokines in response to alloantigen recognition. This suggests potential applications in transplantation medicine for preventing graft rejection .

What are the methodological considerations when using DNA-PKcs antibodies in cancer research?

When utilizing DNA-PKcs antibodies for cancer research, several methodological considerations are essential:

• Expression analysis in tumor tissues:

  • Use validated antibodies for immunohistochemistry on tumor sections

  • Compare expression between tumor and adjacent normal tissue

  • Consider tissue microarrays for high-throughput analysis across multiple samples

  • Correlate expression levels with clinical outcomes (survival, treatment response)

• Activation status assessment:

  • Employ phospho-specific antibodies to evaluate DNA-PKcs activation in tumors

  • Compare phosphorylation patterns between normal and malignant tissues

  • Associate activation status with DNA damage markers (γH2AX, 53BP1)

• Inhibitor response studies:

  • Use phospho-specific antibodies to monitor inhibition of DNA-PKcs activity

  • Assess changes in downstream pathways following inhibitor treatment

  • Combine with functional assays (clonogenic survival, DNA repair capacity)

• Cancer-specific functions:

  • Investigate DNA-PKcs in metastasis by examining its role in angiogenesis and tumor migration

  • Study effects on the tumor microenvironment using co-culture systems

  • Examine regulation of cancer-related genes using ChIP and gene expression analysis

DNA-PKcs has been identified as a metastatic driver in melanoma by stimulating angiogenesis and tumor migration. Upregulation of DNA-PKcs has been observed in multiple cancer types including hepatocellular carcinoma and multiple myeloma, and is associated with radioresistance in thyroid, nasopharyngeal, oral cavity, and cervical cancers .

How can researchers validate the specificity of newly developed DNA-PKcs antibodies?

Rigorous validation is essential when developing or using new DNA-PKcs antibodies. A comprehensive validation strategy should include:

• Western blot validation:

  • Verify detection of a single band at the correct molecular weight (~460 kDa)

  • Test in multiple cell types with known DNA-PKcs expression (HeLa, MCF-7, U2OS)

  • Include negative controls (PRKDC knockout cells, pre-absorption with immunizing antigen)

  • Compare with established antibodies targeting different epitopes

• Genetic validation:

  • Test antibody in wild-type versus CRISPR/Cas9-mediated knockout cells

  • Use siRNA/shRNA knockdown to create a gradient of expression levels

  • Perform rescue experiments with recombinant DNA-PKcs expression

• Phospho-antibody validation:

  • Verify increased signal following DNA damage induction

  • Confirm signal reduction after phosphatase treatment

  • Test specificity using phospho-mimetic and phospho-null mutants

  • Validate with kinase inhibitors to show reduced phosphorylation

• Application-specific validation:

  • Validate separately for each intended application (WB, IP, IF, IHC)

  • Optimize protocols including antigen retrieval methods for IHC

  • Document lot-to-lot variation and optimal conditions

• Cross-reactivity assessment:

  • Test for potential cross-reactivity with related kinases

  • Verify species cross-reactivity if claiming reactivity across species

  • Document specificity across different experimental conditions

A well-validated antibody will show consistent results across multiple validation methods and maintain specificity under varied experimental conditions .

How are DNA-PKcs antibodies being utilized in the development of novel therapeutic approaches?

The development of therapeutic approaches targeting DNA-PKcs is an active area of research, with antibodies playing crucial roles in several aspects:

• Inhibitor development and assessment:

  • Phospho-specific antibodies are used to evaluate inhibitor efficacy

  • Western blots and antibody microarrays help establish structure-activity relationships

  • Immunofluorescence detects subcellular localization changes following inhibitor treatment

  • Pharmacodynamic markers for clinical trials rely on antibody-based detection methods

• Combination therapy evaluation:

  • Antibodies help assess DNA-PKcs status when combining inhibitors with:

    • Radiotherapy (monitoring DNA repair capacity)

    • Chemotherapy (evaluating synergistic effects)

    • Immunotherapy (examining effects on immune responses)

• Companion diagnostics:

  • IHC with DNA-PKcs antibodies can identify patients likely to respond to inhibitors

  • Phospho-specific antibodies may serve as biomarkers of target engagement

  • Expression analysis helps stratify patients for clinical trials

• Beyond inhibition:

  • Novel approaches targeting protein-protein interactions rather than kinase activity

  • Development of proteolysis-targeting chimeras (PROTACs) for DNA-PKcs degradation

  • Potential for antibody-drug conjugates targeting DNA-PKcs in cancer cells

Recently developed inhibitor DA-143 demonstrates enhanced bioavailability compared to earlier compounds, which is critical for effective preclinical and clinical evaluation. This improved solubility facilitates efficacy at reduced doses, addressing a significant limitation of previous DNA-PKcs inhibitors .

What role do DNA-PKcs antibodies play in understanding the relationship between DNA repair and immune function?

DNA-PKcs functions at the intersection of DNA repair and immune responses, making antibodies against it valuable tools for investigating this relationship:

• Viral DNA sensing mechanisms:

  • DNA-PKcs is essential for cGAS/STING-dependent IFN-I responses

  • Antibodies help track DNA-PKcs recruitment to viral DNA

  • Co-localization studies reveal interactions with viral sensing components

  • IP experiments identify complexes formed during immune activation

• T cell signaling pathways:

  • DNA-PKcs regulates critical transcription factors like Egr1 in T cells

  • Phospho-specific antibodies detect DNA-PKcs activation during T cell stimulation

  • ChIP experiments reveal DNA-PKcs association with immune-related gene promoters

  • Inhibitor studies combined with antibody detection demonstrate functional outcomes

• Innate/adaptive immunity intersection:

  • Antibodies help study DNA-PKcs in both innate and adaptive immune contexts

  • Quantitative proteomic approaches with TMT technology identify phosphorylation targets

  • Expression analysis across immune cell types reveals tissue-specific functions

• Therapeutic implications:

  • DNA-PKcs inhibition prevents T cells from producing inflammatory cytokines

  • Potential applications in transplantation medicine to prevent rejection

  • Possible dual targeting of DNA repair and immune function in cancer

Studies have demonstrated that DNA-PKcs functions as a molecular link between DNA damage repair pathways and immune responses, participating in both nuclear DNA repair and cytoplasmic viral DNA sensing. This dual functionality makes it a promising target for therapeutic interventions in both cancer and immune-related disorders .

How can advanced proteomics approaches be combined with DNA-PKcs antibodies for substrate identification?

Integrating DNA-PKcs antibodies with advanced proteomics enables comprehensive identification of substrates and interaction partners:

• Phosphoproteomic approaches:

  • Utilize quantitative mass spectrometry with TMT (tandem mass tag) technology

  • Compare phosphoproteomes between stimulated cells with or without DNA-PKcs inhibition

  • Immunoprecipitate DNA-PKcs to identify associated proteins and phosphorylation targets

  • Employ SILAC labeling for quantitative comparison of substrate phosphorylation

• Experimental design considerations:

  • Include appropriate stimuli (DNA damage, immune activation)

  • Use specific DNA-PKcs inhibitors (NU7441) to distinguish direct from indirect effects

  • Consider temporal dynamics with multiple time points after stimulation

  • Include appropriate controls (kinase-dead mutants, CRISPR knockout cells)

• Validation strategies:

  • Confirm direct phosphorylation using in vitro kinase assays

  • Generate phospho-specific antibodies against identified sites

  • Perform site-directed mutagenesis to assess functional significance

  • Use proximity-based labeling methods (BioID, APEX) to verify spatial relationships

• Functional characterization:

  • Assess how phosphorylation affects substrate localization, stability, or activity

  • Examine cell type-specific differences in substrate utilization

  • Investigate context-dependent (damage, immune stimulation) substrate preferences

A quantitative proteomic mass spectrometry screen utilizing TMT technology identified the transcription factor Egr1 as a DNA-PKcs phosphorylation target in T cell activation. This finding revealed that DNA-PKcs stabilizes Egr1, which is crucial for T cell responses to immunogenic stimuli, suggesting broader influence on immune outcomes than previously understood .

What are the optimal storage and handling conditions for DNA-PKcs antibodies?

Proper storage and handling of DNA-PKcs antibodies is critical for maintaining their performance across applications:

For optimal performance:

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles

• Store antibodies in small aliquots to minimize freeze-thaw cycles

• Some formulations contain preservatives like 0.02% sodium azide and 50% glycerol at pH 7.3

• For small volume antibodies (20µL), aliquoting may be unnecessary for -20°C storage

• Follow manufacturer-specific recommendations as formulations may vary

When handling antibodies during experiments:

• Allow solutions to equilibrate to room temperature before opening

• Centrifuge briefly before use to collect contents at the bottom of the tube

• Use sterile technique when handling reconstituted antibodies

• Return to recommended storage conditions immediately after use

How can researchers troubleshoot common issues with DNA-PKcs antibody applications?

When working with DNA-PKcs antibodies, researchers may encounter several challenges. Here are methodological approaches to troubleshoot common issues:

• Western blot challenges:

  • No signal or weak signal:

    • Increase antibody concentration (start with 1:1000 and adjust as needed)

    • Extend primary antibody incubation time (overnight at 4°C)

    • Use enhanced detection systems for low abundance samples

    • Consider the large size of DNA-PKcs (460 kDa) - extend transfer time

  • Multiple bands:

    • Verify specificity with knockout controls

    • Include protease inhibitors to prevent degradation

    • Use freshly prepared samples to minimize degradation

    • Optimize gel percentage (use low percentage gels for this large protein)

• Immunofluorescence/IHC challenges:

  • High background:

    • Increase blocking time/concentration

    • Optimize antibody dilution (try 1:100-1:500 range)

    • Include additional washing steps

  • No signal:

    • Optimize antigen retrieval (TE buffer pH 9.0 or citrate buffer pH 6.0)

    • Increase antibody concentration

    • Extend primary antibody incubation time

    • Consider signal amplification methods

• Phospho-specific antibody issues:

  • Loss of phospho-signal:

    • Include phosphatase inhibitors in all buffers

    • Process samples quickly and keep cold

    • Verify phosphorylation induction protocol

    • Consider fixation methods that preserve phospho-epitopes

• Cross-reactivity concerns:

  • Validate antibody specificity in your experimental system

  • Include appropriate positive and negative controls

  • Consider blocking with immunizing peptide if available

  • Compare results with alternative antibodies targeting different epitopes

For all applications, appropriate controls are essential: positive controls (HeLa cells, MCF-7 cells), negative controls (knockdown/knockout samples), and procedural controls (secondary antibody only) .

What considerations are important when using DNA-PKcs antibodies in multiplexed imaging or flow cytometry?

When incorporating DNA-PKcs antibodies into multiplexed imaging or flow cytometry protocols, several methodological considerations become critical:

• Panel design considerations:

  • Spectral compatibility:

    • Select fluorophores with minimal spectral overlap

    • Consider brightness hierarchy (assign brightest fluorophores to lowest expressed targets)

    • Plan for appropriate compensation controls

  • Antibody compatibility:

    • Ensure antibodies are from different host species or use directly conjugated antibodies

    • Test for cross-reactivity between antibodies in the panel

    • Verify that detection systems don't interfere with each other

• Staining protocol optimization:

  • Sequential staining:

    • Consider sequential rather than simultaneous staining for complex panels

    • Test different fixation and permeabilization protocols for compatibility

    • Optimize incubation times and temperatures for each antibody

  • Signal-to-noise optimization:

    • Titrate each antibody individually before combining

    • Include appropriate blocking steps between antibody applications

    • Consider signal amplification for low-abundance targets

• Technical considerations for DNA-PKcs detection:

  • Nuclear localization:

    • Ensure permeabilization protocol allows nuclear access

    • Use appropriate nuclear counterstains for localization

    • Consider subcellular fractionation for flow cytometry

  • Phosphorylation detection:

    • Use specific fixation methods that preserve phospho-epitopes

    • Include phosphatase inhibitors throughout the protocol

    • Consider baseline versus activated states in experimental design

• Data analysis approaches:

  • For flow cytometry:

    • Use appropriate gating strategies to analyze nuclear proteins

    • Consider median fluorescence intensity rather than percent positive

    • Include fluorescence minus one (FMO) controls

  • For imaging:

    • Employ quantitative analysis tools for co-localization

    • Use nuclear masks for accurate quantification

    • Consider 3D analysis for volumetric data

These methodological approaches enable researchers to effectively incorporate DNA-PKcs antibodies into complex multiplexed analyses, facilitating the study of DNA-PKcs in relation to other proteins and cellular processes .