RPA4 Antibody

What types of RPA4 antibodies are currently available for research?

Current research laboratories have access to several types of RPA4 antibodies, primarily polyclonal antibodies raised in rabbits or mice. These antibodies target different epitopes, with some specifically binding to the C-terminal region of the protein . Both unconjugated and potentially conjugated versions are available from various suppliers, allowing flexibility in experimental design . Most commercially available RPA4 antibodies have been validated for Western blotting, while some are also suitable for immunoprecipitation, ELISA, immunofluorescence, and immunohistochemistry applications .

What is the expression pattern of RPA4 across different tissues?

RPA4 shows a tissue-specific expression pattern, being preferentially expressed in placental tissue and colon mucosa . This restricted expression profile distinguishes it from the more ubiquitously expressed RPA2 subunit of the canonical RPA complex, suggesting potential tissue-specific functions. When designing experiments targeting endogenous RPA4, researchers should consider these expression patterns to select appropriate cell lines or tissue samples that naturally express detectable levels of the protein .

What are the optimal conditions for using RPA4 antibodies in Western blotting?

For Western blotting applications with RPA4 antibodies, researchers should consider the following methodological approach: use dilutions ranging from 1:200 to 1:1000 depending on the specific antibody and expected expression levels . When preparing samples, whole cell lysates from human cell lines such as HEK293T or HeLa, or mouse tissues like spleen have been successfully used to detect RPA4 . The predicted molecular weight of RPA4 is 29 kDa, though the observed migration pattern may range between 29-32 kDa on SDS-PAGE gels . For optimal results, titrate the antibody concentration for each specific experimental system, as sensitivity can vary between different cellular contexts and protein expression levels.

How should immunoprecipitation experiments with RPA4 antibodies be designed?

For immunoprecipitation of RPA4, researchers should use approximately 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate . HeLa cells have been validated as a suitable source for RPA4 immunoprecipitation . The experimental design should include appropriate controls, such as an isotype-matched negative control antibody and a positive control targeting a known interacting protein. When investigating RPA4's interaction partners, consider the known biology of the alternative RPA complex and its role in DNA repair pathways to guide your experimental approach and interpretation of results.

What are the recommended storage conditions for maintaining RPA4 antibody activity?

To maintain optimal activity of RPA4 antibodies, store them at -20°C in appropriate buffer conditions. Many commercial RPA4 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Under these conditions, the antibodies typically remain stable for one year after shipment without the need for aliquoting . Minimize freeze-thaw cycles to prevent degradation of antibody quality. For working solutions during experiments, keep antibodies on ice and use within the recommended time frame specified by the manufacturer to ensure consistent results.

How can I validate the specificity of my RPA4 antibody?

Validating RPA4 antibody specificity requires a multi-faceted approach. First, confirm the antibody detects a protein of the expected molecular weight (29-32 kDa) in Western blots . Second, compare detection patterns between tissues known to express RPA4 (such as placenta or colon) versus those with minimal expression. Third, consider using RPA4 knockdown or knockout cell lines as negative controls. Fourth, if possible, perform peptide competition assays using the immunizing peptide to confirm binding specificity. Finally, cross-validate results using a second RPA4 antibody targeting a different epitope to ensure consistent detection patterns across different reagents.

What potential cross-reactivity issues should be considered when using RPA4 antibodies?

A significant consideration when working with RPA4 antibodies is potential cross-reactivity with RPA2, which shares 47% sequence identity with RPA4 . This homology can lead to false positive results if antibodies recognize conserved epitopes between these related proteins. To address this concern, researchers should carefully select antibodies that target unique regions of RPA4, particularly if studying systems that express both proteins. Additionally, validation experiments in RPA4-deficient or RPA4-overexpressing systems can help differentiate specific from non-specific signals. When interpreting results, consider the relative expression levels of RPA4 versus RPA2 in your experimental system, as this may influence the apparent specificity of your antibody.

How should I interpret conflicting results between different detection methods using RPA4 antibodies?

When faced with conflicting results between detection methods (e.g., immunofluorescence versus Western blotting), consider several factors: First, different applications expose different epitopes due to variations in protein conformation, fixation, or denaturation. Second, the antibody's affinity may vary across applications, requiring optimization of concentration for each method. Third, the localization and expression level of RPA4 might change under different cellular conditions, affecting detection sensitivity. To resolve discrepancies, validate your findings using multiple antibodies targeting different RPA4 epitopes, employ alternative detection methodologies, and compare your results with published literature on RPA4 biology in similar experimental systems.

How can RPA4 antibodies be utilized to study the switch between canonical and alternative RPA complexes during DNA repair?

Investigating the dynamic switch between canonical (RPA2-containing) and alternative (RPA4-containing) RPA complexes requires sophisticated experimental approaches. Researchers can use RPA4 antibodies in combination with RPA2 antibodies for co-immunoprecipitation studies following DNA damage induction to monitor changes in complex composition . Chromatin immunoprecipitation (ChIP) assays using RPA4 antibodies can track recruitment to DNA damage sites. For advanced applications, consider proximity ligation assays (PLA) to visualize interactions between RPA4 and other repair factors in situ. Time-course experiments following exposure to different DNA-damaging agents can reveal how the balance between these complexes shifts during various repair processes, potentially illuminating functional differences between canonical and alternative RPA complexes.

What methodologies can be employed to study RPA4's role in RAD51-dependent strand exchange and homologous recombination?

To investigate RPA4's functions in RAD51-dependent processes, researchers can implement several approaches using RPA4 antibodies. In vitro reconstitution assays with purified components allow direct assessment of how RPA4-containing complexes influence RAD51 filament formation on ssDNA . For cellular studies, use RPA4 antibodies to immunoprecipitate protein complexes after inducing double-strand breaks, followed by mass spectrometry to identify damage-specific interactors. Combining super-resolution microscopy with RPA4 and RAD51 co-immunostaining can visualize their spatial relationships at repair foci. To assess functional outcomes, measure homologous recombination efficiency using reporter assays in cells with manipulated RPA4 expression levels, correlating results with RPA4 localization detected by your validated antibodies.

How can differential expression of RPA4 in various tissues influence experimental design and data interpretation?

The tissue-specific expression pattern of RPA4, particularly its enrichment in placental and colon mucosa, presents both challenges and opportunities for research . When designing experiments, select cell models that naturally express RPA4 or consider ectopic expression systems for tissues where RPA4 is normally absent. For analysis of clinical samples, use RPA4 antibodies in immunohistochemistry panels to evaluate expression across different tissue types, pathological conditions, or developmental stages. Interpret tissue-specific findings in the context of the unique DNA repair requirements and replication dynamics of each tissue type. Consider that RPA4 might serve specialized functions in highly proliferative tissues with distinctive replication stress profiles, potentially explaining its restricted expression pattern.

What considerations are important when using RPA4 antibodies to study protein-protein interactions within the alternative RPA complex?

When investigating protein-protein interactions involving RPA4, several methodological considerations are critical. First, the epitope recognized by your RPA4 antibody should not interfere with binding surfaces involved in complex formation. For co-immunoprecipitation studies, optimize lysis conditions to preserve native protein complexes while achieving efficient extraction . Consider using crosslinking approaches to capture transient or weak interactions. When analyzing results, account for the competitive relationship between RPA4 and RPA2 for incorporation into the RPA complex . Advanced techniques like biolayer interferometry or surface plasmon resonance with purified components can provide quantitative insights into binding affinities and kinetics of RPA4-containing complexes compared to canonical RPA complexes.

How might RPA4 antibodies contribute to understanding the protein's role in cancer biology?

RPA4's differential expression pattern and its functions in DNA repair pathways suggest potential roles in cancer biology that merit exploration . Researchers can utilize RPA4 antibodies in tissue microarray analyses to evaluate expression across cancer types and correlate findings with clinical outcomes. In mechanistic studies, investigate how altered RPA4 expression affects genome stability, DNA damage response efficiency, and chemotherapy resistance. Compare RPA4 and RPA2 expression ratios in matched normal and tumor samples to determine if the balance between canonical and alternative RPA complexes shifts during oncogenesis. Such studies may reveal whether RPA4 functions as a tumor suppressor or oncogene in different contexts, potentially identifying new therapeutic vulnerabilities in cancers with aberrant DNA repair mechanisms.

What approaches can be used to study post-translational modifications of RPA4 using available antibodies?

Studying post-translational modifications (PTMs) of RPA4 requires specialized experimental approaches. Begin by using general RPA4 antibodies for immunoprecipitation followed by mass spectrometry to identify potential modification sites. Once specific PTMs are identified, develop or obtain modification-specific antibodies. For phosphorylation studies, use phosphatase treatments as controls to confirm specificity of phospho-antibody signals. Employ two-dimensional gel electrophoresis followed by Western blotting to separate differentially modified forms of RPA4. Time-course experiments following cellular stresses can reveal dynamic changes in modification patterns. Compare PTM profiles between RPA4 and the homologous RPA2 protein, which is known to undergo extensive phosphorylation during DNA damage responses, to identify similarities and differences in their regulation.