XRRA1 Antibody

What is XRRA1 and why is it important in radiation research?

XRRA1 (X-ray radiation resistance-associated protein 1) is a protein involved in cellular responses to X-ray radiation . The gene was first identified as being down-regulated by approximately two-fold in an X-ray resistant cell clone (HCT116 Clone2_XRR) compared to control cells . XRRA1 may play an important role in the DNA damage response pathway, with potential implications for understanding cancer radiotherapy responses. Research suggests that XRRA1 might be correlated with cancer development and could be an early expressed gene in response to radiation damage . The protein's high conservation across mammalian species (human, macaque, mouse, rat, pig, and bovine) suggests evolutionarily important functions .

What is the molecular structure and genomic organization of XRRA1?

The human XRRA1 gene is located on chromosome 11q13.3 and consists of 11 exons spanning approximately 64 kb . The human XRRA1 cDNA is 1987 nucleotides long, encoding a protein of 559 amino acids . The exon-intron junctions of the XRRA1 gene follow the Kozak consensus sequence, and these junctions are well-conserved across human, mouse, and rat species . Alternative splicing has been documented, with at least one splice variant identified that lacks exon three . The protein structure includes functional domains that support its role in radiation response, though detailed structural analysis continues to be an active area of research.

What is the tissue expression pattern of XRRA1?

XRRA1 is expressed in multiple tissues, with predominant expression observed in the testis of both human and macaque . cDNA microarray analysis has shown three-fold higher expression of XRRA1 in macaque testis relative to other tissues . The protein is also expressed in most tumor cell types . Interestingly, mouse Xrra1 has been detected in mouse embryonic stem cells R1, suggesting potential roles in early development . XRRA1's differential expression pattern across normal and cancer tissues provides important context for antibody-based studies examining its function and regulation.

How does XRRA1 contribute to radiation resistance mechanisms?

XRRA1's role in radiation resistance appears to be linked to DNA damage response pathways. Research has shown differential expression of XRRA1 after X-radiation in HCT116 clones with significantly different X-radiation responses . The protein may interact with key components of DNA repair mechanisms. One intriguing finding is that COS-7 cells over-expressing XRRA1 showed evidence of lacking Ku86 (also known as Ku80 or XRCC5), a critical non-homologous end joining (NHEJ) DNA repair molecule, in the nucleus . This suggests XRRA1 might modulate DNA repair efficiency through interactions with established repair factors. The exact molecular mechanisms by which XRRA1 influences radiation resistance warrant further investigation using genetic knockout studies, protein interaction analyses, and functional recovery experiments.

What are the challenges in detecting endogenous versus overexpressed XRRA1?

Detection of endogenous XRRA1 presents several challenges due to its relatively low expression levels in many cell types compared to its overexpressed form. When using XRRA1 antibodies, researchers should consider the following factors: (1) The predicted band size for human XRRA1 is approximately 90 kDa by Western blot analysis , though actual observed sizes may vary due to post-translational modifications; (2) Subcellular localization studies show that GFP-XRRA1 fusion protein can be detected in both the nucleus and cytoplasm of HCT116 clones and COS-7 cells , requiring optimization of cell fractionation protocols; (3) Differences in antibody sensitivities between applications (Western blot versus immunohistochemistry) necessitate validation for each experimental approach. Researchers should include appropriate positive controls (such as XRRA1-overexpressing cells) and negative controls when establishing detection protocols.

How might XRRA1 interact with other DNA damage response factors?

XRRA1's potential interactions with DNA damage response factors represent a complex research question. The observation that XRRA1 overexpression affects nuclear localization of Ku86 suggests it may influence the recruitment or retention of DNA repair factors at damage sites. This interaction could involve direct protein-protein binding or indirect effects through signaling cascades. The relationship between XRRA1 and the ATM kinase pathway, a central mediator of radiation response that "mediates the prompt induction of various signaling pathways" , remains to be fully characterized. Methodological approaches to investigate these interactions should include co-immunoprecipitation studies, proximity ligation assays, and functional complementation experiments in cells with modulated XRRA1 expression.

What are the optimal conditions for using XRRA1 antibodies in Western blotting?

For optimal Western blotting detection of XRRA1, researchers should consider the following protocol elements: (1) Use fresh cell or tissue lysates with complete protease inhibitor cocktails to prevent protein degradation; (2) Load adequate protein amounts (typically 20-50 μg per lane) to ensure detection of potentially low-abundance XRRA1; (3) Employ a dilution ratio of 1/1000 for commercially available XRRA1 antibodies as demonstrated in published protocols ; (4) Include positive control samples such as HepG2, HL-60, or U-251 MG cell lysates, which have been validated for XRRA1 detection ; (5) Be aware of the predicted band size of approximately 90 kDa, though multiple bands may represent splice variants or post-translationally modified forms . For signal development, secondary antibody (such as goat polyclonal to Rabbit IgG) at 1/10000 dilution has shown effective results in published studies .

How should immunohistochemistry protocols be optimized for XRRA1 detection?

For successful immunohistochemical detection of XRRA1 in tissue samples, researchers should follow these methodological guidelines: (1) Use paraffin-embedded tissue sections with proper antigen retrieval methods, typically heat-induced epitope retrieval in citrate buffer (pH 6.0); (2) Apply XRRA1 antibody at a dilution of 1/100 as validated in human small intestine tissue ; (3) Include tissue-specific positive controls with known XRRA1 expression (testis tissue would be optimal based on expression data) ; (4) Employ appropriate blocking solutions to minimize background staining; (5) Consider testing multiple antibody incubation times and temperatures to optimize signal-to-noise ratio; (6) Validate specificity through peptide competition assays or comparison with other detection methods. Researchers should be aware that XRRA1 localization may be both nuclear and cytoplasmic , requiring careful evaluation of staining patterns.

What experimental approaches are recommended for studying XRRA1 function in radiation response?

To investigate XRRA1's functional role in radiation response, the following experimental approaches are recommended: (1) Gene knockdown/knockout studies using siRNA, shRNA, or CRISPR-Cas9 systems followed by assessment of cellular radiosensitivity; (2) Overexpression studies using GFP-tagged XRRA1 constructs to examine localization before and after radiation exposure ; (3) Time-course analysis of XRRA1 expression following various radiation doses to establish dose-response relationships; (4) Examination of interactions with known DNA repair factors such as Ku86 using co-immunoprecipitation or proximity ligation assays ; (5) Measurement of DNA damage repair kinetics (using γH2AX foci or comet assays) in cells with modulated XRRA1 expression; (6) Comparison of different radiation types (X-ray, gamma, UV) to determine specificity of XRRA1's role. These approaches should be conducted in relevant cell models, particularly those with established radiation response profiles such as the HCT116 clone series .

XRRA1 Antibody Applications and Validation Status

XRRA1 Expression Across Cell Types

Genomic Characteristics of XRRA1 Across Species

What are common challenges when using XRRA1 antibodies and how can they be resolved?

Researchers working with XRRA1 antibodies commonly encounter several challenges that require specific troubleshooting approaches. For weak or absent Western blot signals, consider: (1) Increasing protein loading amounts; (2) Optimizing antibody concentration beyond the standard 1/1000 dilution ; (3) Extending primary antibody incubation time to overnight at 4°C; (4) Using enhanced chemiluminescence detection systems with longer exposure times; (5) Verifying sample integrity through detection of housekeeping proteins. For high background in immunohistochemistry: (1) Increase blocking time and concentration; (2) Optimize antibody dilution starting from the recommended 1/100 ; (3) Include additional washing steps; (4) Test alternative antigen retrieval methods. For inconsistent results between experiments: (1) Standardize lysate preparation methods; (2) Use freshly prepared buffers; (3) Include positive control samples (HepG2, HL-60, or U-251 MG lysates ) in each experiment; (4) Consider lot-to-lot variability in antibodies and keep detailed records of antibody sources.

How should researchers interpret complex XRRA1 expression patterns?

Interpretation of XRRA1 expression patterns requires careful consideration of several factors. The protein's dual nuclear and cytoplasmic localization means that changes in distribution between compartments may be physiologically significant, particularly following radiation exposure. When analyzing Western blot results, researchers should note that: (1) Multiple bands may represent splice variants, including the documented variant lacking exon three ; (2) Post-translational modifications might alter apparent molecular weight from the predicted 90 kDa ; (3) Cross-reactivity with related proteins should be excluded through peptide competition assays. For immunohistochemistry or immunofluorescence interpretation: (1) Compare staining patterns with both positive and negative controls; (2) Correlate XRRA1 expression with functional outcomes such as radiation sensitivity; (3) Consider co-localization with DNA repair factors such as Ku86 for mechanistic insights. In all cases, validation with multiple antibodies or complementary techniques (such as mRNA quantification) strengthens interpretation.

What emerging techniques might advance our understanding of XRRA1 function?

Emerging research techniques offer promising avenues for advancing our understanding of XRRA1 function. CRISPR-Cas9 gene editing allows precise manipulation of XRRA1 genomic sequences to create knockout cell lines or introduce specific mutations for structure-function analysis. Live-cell imaging using fluorescently tagged XRRA1 can reveal dynamic changes in localization and interactions following radiation exposure in real-time. Proximity-based labeling approaches (BioID or APEX) could identify novel protein interaction partners within the cellular microenvironment. Single-cell techniques may reveal heterogeneity in XRRA1 expression and function within populations of cancer cells, potentially explaining differential radiation responses. Proteomics approaches focusing on post-translational modifications of XRRA1 could identify regulatory mechanisms controlling its activity. These advanced methodologies, combined with appropriate XRRA1 antibodies and expression systems, represent the frontier of research into this radiation response protein.

How might XRRA1 research contribute to clinical applications in cancer radiotherapy?

XRRA1 research has potential implications for improving cancer radiotherapy outcomes. The protein's role in radiation resistance mechanisms suggests it could serve as a biomarker for predicting tumor radiotherapy response. Methodological approaches for clinical translation include: (1) Immunohistochemical analysis of XRRA1 expression in pre-treatment tumor biopsies correlated with clinical outcomes; (2) Development of XRRA1 expression or activity assays that could guide radiation dose personalization; (3) Investigation of pharmacological modulators of XRRA1 activity that might overcome radiation resistance; (4) Combination studies of XRRA1 targeting with standard radiotherapy protocols. The finding that XRRA1 might affect Ku86 localization suggests potential synergies with DNA repair inhibitors. Researchers should focus on validating XRRA1 as a predictive biomarker across diverse tumor types and establishing standardized detection protocols suitable for clinical implementation.