Prkdc Antibody

What is PRKDC and why is it important in research?

PRKDC, also known as DNA-PKcs, HYRC, HYRC1, DNPK1, and p460, belongs to the PI3/PI4-kinase family. It functions as a serine/threonine-protein kinase that acts as a molecular sensor for DNA damage. PRKDC is critically involved in DNA nonhomologous end joining (NHEJ), required for double-strand break (DSB) repair and V(D)J recombination. It must bind to DNA to express its catalytic properties and promotes processing of hairpin DNA structures in V(D)J recombination by activating the hairpin endonuclease artemis (DCLRE1C) . Recent research has identified PRKDC as both a predictive biomarker and potential drug target for immune checkpoint inhibitors .

What are the key characteristics of PRKDC protein?

PRKDC has a calculated molecular weight of 469 kDa, though its observed molecular weight in experimental settings ranges from 350-460 kDa . The protein is encoded by the gene with GenBank Accession Number NM_006904 and has UNIPROT ID P78527 . PRKDC functions as a catalytic subunit of DNA-dependent protein kinase and must be bound to DNA to express its catalytic properties . It plays essential roles in DNA repair mechanisms, particularly nonhomologous end joining.

What types of PRKDC antibodies are available for research?

Commercial PRKDC antibodies are available in various formats, including rabbit polyclonal antibodies like the 19983-1-AP that has been tested for reactivity with human samples and cited for reactivity with human, mouse, and rat samples . These antibodies are typically available in liquid form, purified through antigen affinity methods, and stored in buffers containing PBS with preservatives such as sodium azide and glycerol .

Which applications can PRKDC antibodies be used for?

PRKDC antibodies have been validated for multiple applications including Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), Immunoprecipitation (IP), Co-Immunoprecipitation (CoIP), and ELISA applications . According to published literature, these antibodies have been successfully used in at least 19 publications for WB, 3 publications for IHC, and 1 publication each for IF and CoIP . This versatility makes PRKDC antibodies valuable tools in various experimental approaches.

What are the recommended protocols for Western Blot using PRKDC antibodies?

For Western Blot applications, PRKDC antibodies are typically used at dilutions ranging from 1:500 to 1:3000 . The high molecular weight of PRKDC (350-460 kDa observed) requires special considerations during electrophoresis, including using low percentage gels (typically 6-8%) and longer running times. For protein extraction, RIPA buffer supplemented with protease and phosphatase inhibitors at 4°C is effective, as demonstrated in published protocols . Standard detection methods using ECL-Plus detection systems are suitable for visualization .

How should immunoprecipitation experiments with PRKDC antibodies be designed?

For immunoprecipitation applications, the recommended protocol involves using 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate . Published research has confirmed successful IP detection in HeLa cells . When designing co-immunoprecipitation experiments to study protein-protein interactions with PRKDC, researchers should consider using gentle lysis buffers that preserve protein complexes while ensuring sufficient extraction of this large nuclear protein.

What considerations should be made when designing IHC experiments with PRKDC antibodies?

When performing immunohistochemistry with PRKDC antibodies, appropriate antigen retrieval methods are crucial, typically using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) due to formalin fixation effects on protein epitopes. Several studies have successfully used PRKDC antibodies for IHC applications , particularly in cancer tissue samples. Researchers should optimize blocking conditions to minimize background staining and consider counterstaining to provide structural context.

How can researchers verify the specificity of PRKDC antibody signals?

Verifying antibody specificity is critical for reliable results. Researchers should include appropriate positive controls (such as HeLa or MCF-7 cells, which have been tested positive for PRKDC detection) . Negative controls should include PRKDC knockout samples or siRNA-treated samples when possible. Western blot analysis should confirm a band at the expected molecular weight range of 350-460 kDa . Additionally, comparing results across multiple detection methods can provide stronger evidence of specificity.

How should researchers interpret PRKDC expression data in cancer research?

When interpreting PRKDC expression data in cancer research, it's important to consider both protein levels and mRNA expression. Higher PRKDC expression has been significantly associated with basal-like PAM50 subtype and ER negativity in breast cancer . Additionally, high PRKDC expression correlates with poor disease-free survival rates in multiple cancer datasets . Researchers should analyze expression data in context with other biomarkers and clinical parameters for comprehensive interpretation.

What is the significance of PRKDC mutations in cancer and immunotherapy response?

PRKDC mutations have emerged as potential biomarkers for immunotherapy response. Research has shown that among patients whose tumors harbored PRKDC mutations, 75%, 53.8%, and 50% of those with lung cancer, melanoma, and renal cell carcinoma, respectively, responded to immunotherapy . These mutations are often truncating and located in functional domains or destabilizing regions of the PRKDC protein structure . PRKDC mutations are also significantly associated with high mutation load in several cancer types and with microsatellite instability-high status in gastric and colon cancers .

How can PRKDC antibodies be used to study DNA damage response pathways?

PRKDC antibodies are valuable tools for studying DNA damage response pathways, particularly nonhomologous end joining (NHEJ). Researchers can use these antibodies to monitor PRKDC recruitment to DNA damage sites through immunofluorescence techniques, often in conjunction with other DNA damage markers like γH2A.X . Co-immunoprecipitation experiments with PRKDC antibodies can identify interaction partners in the DNA repair complex. Phospho-specific antibodies can be used to monitor PRKDC activation state following DNA damage induction by radiation or chemotherapeutic agents.

What approaches can be used to study PRKDC's role as a biomarker in immunotherapy response?

To investigate PRKDC's potential as a biomarker for immunotherapy response, researchers can employ multiple approaches. Immunohistochemical staining of patient tumor samples can evaluate PRKDC protein expression levels before and after treatment . Next-generation sequencing can identify PRKDC mutations that correlate with response . Animal models with PRKDC knockout or inhibition, such as the CT26 model described in the literature, can be used to evaluate enhanced efficacy of immune checkpoint inhibitors . Combination of PRKDC status with other biomarkers like microsatellite instability and mutation load can provide a more comprehensive prediction model.

How can researchers use PRKDC antibodies in combination with other biomarkers?

Researchers studying cancer biomarkers can use PRKDC antibodies in multiplex immunofluorescence or immunohistochemistry to simultaneously detect multiple markers in tissue samples. This approach has been valuable in breast cancer research, where combining DNA-PKcs IHC assessment with key immune biomarkers provides insight into basal-like heterogeneity . Correlation of PRKDC protein levels with mRNA expression data from techniques like RNA-seq can validate findings across different analytical platforms. Researchers should consider standardized protocols and scoring systems for consistent biomarker evaluation.

What are common challenges when detecting PRKDC in Western blots?

The high molecular weight of PRKDC (350-460 kDa) presents several challenges in Western blot detection. Researchers often encounter difficulties with complete protein transfer from gel to membrane, particularly with standard semi-dry transfer systems. Using wet transfer systems with extended transfer times (overnight at low voltage) and lower percentage SDS-PAGE gels (6-8%) can improve results . Degradation during sample preparation can lead to multiple bands or smearing; using fresh samples with complete protease inhibitor cocktails is recommended. For certain applications, gradient gels may provide better resolution.

How can researchers optimize immunoprecipitation experiments with PRKDC antibodies?

Optimizing immunoprecipitation of PRKDC requires careful consideration of several factors. The large size of PRKDC may necessitate adjustments to standard IP protocols, including longer incubation times with antibodies (overnight at 4°C). Researchers should titrate antibody amounts (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate is recommended) . Pre-clearing lysates can reduce non-specific binding. When performing co-IP experiments, gentler lysis conditions may be necessary to preserve protein-protein interactions, though this must be balanced with efficient extraction of this nuclear protein.

What controls should be included when using PRKDC antibodies for DNA damage research?

When using PRKDC antibodies in DNA damage research, appropriate controls are essential for reliable interpretation. Positive controls should include samples treated with known DNA-damaging agents (e.g., ionizing radiation, etoposide, or bleomycin). Negative controls should include PRKDC knockout or knockdown samples when possible . Time-course experiments following DNA damage induction can capture dynamic changes in PRKDC localization or activation. Including other DNA damage markers such as γH2A.X provides internal validation, as demonstrated in PRKDC knockout studies where higher γH2A.X levels were observed compared to parental cells .

How might PRKDC inhibition be combined with immunotherapy in cancer treatment?

Emerging research suggests promising directions for combining PRKDC inhibition with immunotherapy. Animal studies have demonstrated that PRKDC knockout or DNA-PK inhibitor treatment enhanced the efficacy of anti-PD-L1 antibody treatment in the CT26 animal model . Future research should explore optimal dosing and timing of combination therapies, as well as identifying cancer types most likely to benefit from this approach. Investigation of mechanisms underlying the synergy between PRKDC inhibition and checkpoint blockade could reveal additional therapeutic targets. Clinical trials evaluating safety and efficacy of these combinations in patients represent an important next step.

What novel techniques might enhance PRKDC antibody-based research?

Several emerging techniques hold promise for advancing PRKDC antibody-based research. Mass cytometry (CyTOF) could enable simultaneous detection of PRKDC along with dozens of other proteins at the single-cell level. Proximity ligation assays could provide superior sensitivity for detecting PRKDC interactions with other proteins in situ. Super-resolution microscopy techniques could reveal detailed spatial organization of PRKDC at DNA damage sites. Development of antibodies recognizing specific phosphorylation states of PRKDC could provide deeper insights into its activation mechanisms in different contexts.

How can researchers integrate PRKDC protein data with genomic and transcriptomic information?

Integrating multi-omics data is increasingly important in PRKDC research. Researchers can correlate PRKDC protein levels (detected via antibody-based methods) with PRKDC mRNA expression data from RNA-seq or microarray studies . Mapping PRKDC mutations identified through next-generation sequencing to protein domains can provide insights into structure-function relationships . Single-cell multi-omics approaches could reveal heterogeneity in PRKDC expression and mutation status within tumors. Integration of these data types with clinical outcomes can strengthen biomarker development and reveal new biological insights about PRKDC's roles in health and disease.