Recombinant Chicken AT-rich interactive domain-containing protein 5B (ARID5B), partial

What is the structural composition of Chicken AT-rich interactive domain-containing protein 5B?

Chicken ARID5B contains an AT-rich interactive domain that preferentially binds to AT-rich DNA sequences, similar to other ARID-containing proteins. The protein functions primarily as a transcriptional regulator, with the ARID domain mediating interactions with specific genomic regions. Like other homeodomain-containing proteins, ARID5B target sequences are characteristically AT-rich, which is consistent with its binding specificity and regulatory functions . The protein can be expressed in both full-length and partial formats depending on experimental requirements, with each variant potentially exhibiting different binding characteristics or domain-specific functions .

How do researchers validate the authenticity of recombinant Chicken ARID5B?

Authentication of recombinant Chicken ARID5B typically involves multiple validation techniques. Based on methodologies used for similar proteins, researchers should employ co-immunoprecipitation with commercial anti-ARID5B antibodies to confirm protein identity . Western blot analysis using alkaline-phosphatase detection systems and commercially available anti-ARID5B polyclonal antibodies provides additional verification . Functional validation through DNA-binding assays is also essential, as ARID5B should demonstrate specificity for AT-rich sequence regions, similar to other homeodomain proteins . Protein purity should exceed 80% as determined by SDS-PAGE, and endotoxin levels should be maintained below 1.0 EU per μg of protein using the LAL method to ensure experimental reliability .

What expression systems are optimal for producing recombinant Chicken ARID5B?

For optimal expression of recombinant Chicken ARID5B, mammalian cell systems are frequently employed, particularly when post-translational modifications are critical for functional studies . When using bacterial expression systems such as E. coli BL21 (DE3), researchers should consider strategies to minimize protein aggregation, including cultivation at reduced temperatures (16°C instead of 37°C) and using lower IPTG concentrations (0.2 mM) for extended induction periods (approximately 18 hours) . For protein purification, His-tagged recombinant ARID5B can be effectively isolated using Ni-NTA affinity chromatography, with optimized buffer conditions: wash buffer containing 50 mM phosphate, 0.3 M NaCl, and 20 mM imidazole (pH 8.0), followed by elution with 50 mM phosphate, 0.5 M NaCl, and 0.5 M imidazole (pH 8.0) . This approach yields purified protein suitable for downstream applications in experimental research.

What are the performance parameters of ELISA-based detection for Chicken ARID5B?

The Chicken ARID5B ELISA assay offers high specificity and sensitivity for quantitative detection. Technical specifications indicate a detection range of 5.0-100 μg/ml with sensitivity exceeding 1.0 μg/ml . Precision analysis demonstrates excellent reproducibility, with standard deviation less than 8% for standards repeated 20 times on the same plate, and less than 10% when the same sample is measured 20 times by different operators . The assay shows negligible cross-reactivity or interference with ARID5B analogues, making it suitable for complex biological samples . For optimal results, researchers should validate each lot of reagents using standard curves and appropriate positive and negative controls, especially when working with novel sample types or modified experimental conditions.

How can researchers optimize sample preparation for ARID5B detection in various tissue types?

Optimization of sample preparation for ARID5B detection requires tissue-specific considerations. For brain tissue samples, researchers can adapt the protocol described for similar proteins: deparaffinize tissue sections in xylene, hydrate with graded alcohol, and perform antigen retrieval with EDTA buffer (1.0 M, 0.05% Tween-20) at 95–100°C . For blocking, normal horse serum at 37°C in a humidified chamber prevents non-specific binding. When working with homogenized tissues, extraction should be performed in cold lysis buffer (10 mM Tris–HCl, 0.3 M NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, pH 7.5) followed by centrifugation at 12,000× g for 20 minutes to obtain clear supernatant . For complex matrices, validation of extraction efficiency is essential by testing recovery rates using spiked samples containing known amounts of recombinant Chicken ARID5B.

What advanced immunological approaches are available for studying Chicken ARID5B interactions?

For investigating Chicken ARID5B protein interactions, researchers can employ magnetic bead-based immunoprecipitation techniques. Pre-coupled magnetic beads conjugated with recombinant ARID5B (similar to those available for mouse ARID5B) provide a powerful tool for capturing binding partners with high specificity . These uniformly-sized magnetic beads with large surface area enable convenient and rapid capture of target molecules while maintaining high specificity . The technique is particularly valuable for pull-down assays to identify novel protein-protein interactions. For protein-DNA interaction studies, researchers should consider electrophoretic mobility shift assays and DNase I footprinting techniques, which have been successfully used for similar AT-rich domain-containing proteins to determine sequence-specific DNA binding . These approaches can identify consensus binding sequences and characterize binding affinities of ARID5B to various DNA motifs.

How should researchers design experiments to investigate ARID5B's role in transcriptional regulation?

When investigating ARID5B's transcriptional regulatory functions, researchers should design experiments that examine both DNA binding specificity and transcriptional activation capability. Based on studies with similar AT-rich domain proteins, researchers can isolate DNA fragments containing ARID5B binding sites using ARID5B-fusion proteins (such as ARID5B-GST) . Both random oligonucleotides and genomic DNA can serve as sources for identifying binding sequences. Following isolation, DNA fragments should be sequenced and tested in binding assays to determine affinity variations. To determine specific binding sequences, DNase I footprinting should be performed on the isolated genomic fragments . For functional validation of ARID5B's transcriptional activity, identified genomic target sequences can be introduced into reporter plasmids and tested in ARID5B-expressing cells to assess transcriptional enhancement . This comprehensive approach will help elucidate both the binding preferences and transcriptional regulatory functions of Chicken ARID5B.

What considerations are critical when designing co-immunoprecipitation studies with Chicken ARID5B?

Co-immunoprecipitation studies with Chicken ARID5B require careful consideration of experimental conditions to maintain protein-protein interactions while minimizing non-specific binding. Based on protocols for similar proteins, researchers should prepare cell or tissue lysates in gentle lysis buffers containing appropriate protease inhibitors (such as 1 mM PMSF) and stabilizing agents . When using recombinant ARID5B for pull-down assays, authenticity validation is essential through preliminary immunoprecipitation with commercial anti-ARID5B antibodies . For identifying novel interaction partners, mass spectrometry techniques such as LC-MS/MS should be employed to analyze co-precipitated proteins . Control experiments using non-specific antibodies or lysates from cells not expressing ARID5B are crucial for distinguishing genuine interactions from background binding. Researchers should also consider crosslinking approaches for capturing transient interactions and varying salt concentrations to establish interaction strength. For verification of identified partners, reciprocal co-immunoprecipitation and biophysical interaction assays provide additional validation.

What are the key methodological differences when working with partial versus full-length Chicken ARID5B?

When working with partial versus full-length Chicken ARID5B, researchers must account for significant methodological differences throughout the experimental workflow. Expression efficiency may vary considerably between truncated and complete protein constructs, with partial constructs often showing improved expression due to reduced complexity and lower propensity for aggregation . For recombinant production, both versions require custom synthesis with lead times between 5-9 weeks , but optimization parameters (temperature, inducer concentration, and cultivation time) should be independently established for each construct. Functional characterization will reveal distinct properties: partial constructs containing only the ARID domain may demonstrate robust DNA binding but lack protein-protein interaction domains present in the full-length version, potentially affecting complex formation and transcriptional regulation capabilities. For interaction studies, full-length ARID5B provides a comprehensive platform for identifying all potential binding partners, while domain-specific constructs help pinpoint interaction regions with greater precision. Storage stability may also differ, requiring protein-specific optimization of buffer conditions and temperature to maintain activity.

What experimental approaches can distinguish between ARID5B isoforms in chicken tissues?

To distinguish between potential ARID5B isoforms in chicken tissues, researchers need to implement a multi-faceted experimental approach. RT-PCR with isoform-specific primers designed to span potential splice junctions represents the foundation for identifying transcript variants. For protein-level discrimination, researchers should develop isoform-specific antibodies targeting unique epitopes or employ high-resolution techniques like 2D gel electrophoresis coupled with Western blotting to separate proteins based on both molecular weight and isoelectric point differences. Mass spectrometry-based proteomics offers the highest resolution for identifying isoform-specific peptides in complex samples. When studying recombinant versions of different isoforms, functional discrimination through DNA-binding assays will reveal potential differences in sequence preferences or binding affinities . Additionally, transcriptional activation assays using reporter constructs can identify functional differences between isoforms in various cellular contexts. Finally, tracking tissue-specific expression patterns through quantitative PCR or immunohistochemistry will provide insights into the biological significance of each isoform in different chicken tissues.

How can researchers investigate ARID5B's role in host-pathogen interactions in avian species?

The investigation of ARID5B's potential role in host-pathogen interactions in avian species represents an emerging research direction, drawing from methodologies used in studies of similar proteins. Building on the observation that SEPT5 is upregulated in H5N1-infected chicken brains , researchers should first examine whether ARID5B expression is similarly altered during viral infection. This can be accomplished through quantitative PCR and Western blot analysis of infected versus uninfected tissues. Immunohistochemistry using anti-ARID5B antibodies on tissue sections from infected and control chickens, following antigen retrieval with EDTA buffer at 95-100°C, will reveal spatial distribution changes during infection . To identify potential pathogen-related binding partners, recombinant ARID5B can be used for co-immunoprecipitation with lysates from infected tissues, followed by LC-MS/MS analysis to identify interacting proteins . For functional validation, researchers can employ gene silencing or overexpression approaches in cell culture models of infection to assess ARID5B's impact on viral replication and host cell responses. These comprehensive approaches will provide insights into whether ARID5B plays a role in avian immune responses or is manipulated during pathogen invasion.

What methodological approaches can reveal ARID5B's role in chromatin remodeling?

Investigating ARID5B's role in chromatin remodeling requires sophisticated methodologies that capture protein-DNA interactions within the nuclear environment. ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) represents the gold standard for mapping ARID5B binding sites genome-wide in chicken cells. This technique requires highly specific anti-ARID5B antibodies or expression of tagged ARID5B constructs, followed by crosslinking, chromatin fragmentation, immunoprecipitation, and next-generation sequencing. ATAC-seq (Assay for Transposase-Accessible Chromatin with sequencing) can complement ChIP-seq by identifying regions where ARID5B binding correlates with changes in chromatin accessibility. For mechanistic studies, researchers should employ nucleosome remodeling assays using reconstituted chromatin templates and purified recombinant ARID5B to assess direct effects on nucleosome positioning or stability. Co-immunoprecipitation with known chromatin remodeling complexes will identify potential collaborating factors. Hi-C or similar chromosome conformation capture techniques can reveal how ARID5B influences three-dimensional genome organization. Finally, targeted epigenetic profiling of ARID5B-bound regions will determine if its binding correlates with specific histone modifications, providing insights into its functional impact on the epigenetic landscape in chicken cells.

What are the critical parameters for developing CRISPR-Cas9 approaches to study ARID5B function in chicken cells?

Developing CRISPR-Cas9 approaches for studying ARID5B function in chicken cells requires careful consideration of several critical parameters to ensure efficient gene editing and meaningful functional analysis. The design of guide RNAs (gRNAs) should target conserved functional domains of ARID5B, particularly the AT-rich interactive domain, while avoiding regions with high homology to other ARID family members. Researchers should design multiple gRNAs targeting different exons and validate their efficiency using T7 Endonuclease I assay or next-generation sequencing. For delivery into chicken cells, optimized transfection protocols must be established for each cell type, with particular attention to primary chicken cells which may require specialized conditions. When generating knockout cell lines, researchers should comprehensively characterize the edited cell lines through sequencing, RT-PCR, and Western blotting to confirm the absence of functional ARID5B. For knock-in approaches (introducing tagged versions or specific mutations), the design of homology-directed repair templates requires careful consideration of homology arm length and position. Phenotypic analysis should include transcriptomic profiling (RNA-seq), DNA-binding assays (ChIP-seq), and functional readouts relevant to ARID5B's presumed role in transcriptional regulation. Off-target effects should be systematically assessed through computational prediction followed by targeted sequencing of potential off-target sites.