At5g35735 Antibody

What is At5g35735 and what is its function in Arabidopsis thaliana?

At5g35735 is a protein-coding gene in Arabidopsis thaliana (Mouse-ear cress) with UniProt accession number Q9FKH6 . Based on current research, this protein appears to be involved in cellular regulatory networks that mediate plant responses to pathogen infection. The protein may function within signal transduction pathways that are activated during host-pathogen interactions, potentially contributing to the plant's defense mechanisms against pathogens such as Salmonella . The specific molecular function involves protein-protein interactions within larger regulatory networks that influence gene expression patterns during infection processes.

What detection methods are available for studying At5g35735 protein expression?

Multiple approaches can be employed to study At5g35735 protein expression:

• Western blotting: Using the At5g35735 antibody (e.g., CSB-PA194840XA01DOA) for protein detection in tissue extracts

• Immunohistochemistry: For localization studies in plant tissues

• Protein interaction studies: Including co-immunoprecipitation and pull-down assays

• Protein-DNA interaction analysis: For investigating potential transcription factor activity

When selecting detection methods, researchers should consider the cellular localization of At5g35735 and potential cross-reactivity with related proteins in Arabidopsis.

How does At5g35735 relate to plant immunity pathways?

Current research suggests At5g35735 may participate in signaling networks activated during pathogen response. Similar to other proteins identified in Salmonella infection studies, At5g35735 could be part of the complex interplay between various biomolecules regulating cellular behavior during infection . The protein may function within cascades that ultimately influence transcription factor activity and the expression of defense-related genes. Understanding its role requires integration with broader studies of plant immunity pathways and transcriptional regulation networks.

How can At5g35735 antibodies be used to study host-pathogen interactions?

At5g35735 antibodies enable sophisticated research approaches for investigating host-pathogen interactions:

• Signal transduction analysis: Track changes in At5g35735 protein levels during different stages of infection using quantitative Western blotting

• Protein complex identification: Use antibody-based affinity purification followed by mass spectrometry to identify interaction partners during infection

• Pathogen-induced relocalization: Employ immunolocalization to detect potential changes in At5g35735 cellular distribution following pathogen exposure

• Chromatin dynamics: If At5g35735 functions as a transcription factor, ChIP (Chromatin Immunoprecipitation) assays can map its DNA binding sites during infection

These approaches can be integrated into systems biology frameworks similar to those used in SDREM (Signaling and Dynamic Regulatory Events Miner) analyses of Arabidopsis responses to Salmonella infection .

What role might At5g35735 play in transcriptional regulation during infection?

Research on host-pathogen interactions suggests At5g35735 could function as a master regulator (MR) within transcriptional networks activated during infection. Similar transcription factors identified in Arabidopsis have been shown to regulate gene clusters with similar expression profiles in response to Salmonella infection . To investigate this:

• Compare At5g35735 binding domains with known transcription factors

• Analyze DNA-binding motifs and potential target genes

• Correlate At5g35735 expression with gene clusters showing similar expression patterns during infection

• Validate direct regulatory relationships through ChIP-seq or similar approaches

The protein might function similarly to transcription factors identified in STEM (Short Time-series Expression Miner) clusters during Salmonella infection, coordinating expression of defense-related genes .

How does At5g35735 compare to related proteins in the Arabidopsis proteome?

When studying At5g35735, researchers should consider its relationship to other Arabidopsis proteins:

This comparative approach is important for validating antibody specificity and for contextualizing At5g35735 function within larger protein families or functional groups .

What are the optimal protocols for using At5g35735 antibodies in experimental systems?

When designing experiments with At5g35735 antibodies, consider these methodological recommendations:

For Western blotting:

• Sample preparation: Extract proteins using buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% NP-40, and protease inhibitors

• Protein separation: Use 10-12% SDS-PAGE gels

• Transfer conditions: 100V for 60-90 minutes using PVDF membrane

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature

• Primary antibody: Dilute At5g35735 antibody 1:1000 in blocking solution

• Detection: Use HRP-conjugated secondary antibody and ECL detection system

For immunolocalization:

• Fixation: 4% paraformaldehyde in PBS for 20 minutes

• Permeabilization: 0.1% Triton X-100 for 10 minutes

• Blocking: 2% BSA in PBS for 30 minutes

• Primary antibody: Incubate with At5g35735 antibody (1:500) overnight at 4°C

• Visualization: Fluorophore-conjugated secondary antibody with appropriate filters

Always include positive and negative controls, and validate antibody specificity using knockout lines or competing peptides when available.

How should researchers validate At5g35735 antibody specificity?

Antibody validation is crucial for generating reliable data. Implement these validation strategies:

• Genetic controls: Compare antibody signal between wild-type and At5g35735 knockout/knockdown plants

• Peptide competition: Pre-incubate antibody with immunizing peptide to confirm signal specificity

• Cross-species validation: Test reactivity against homologous proteins from related plant species

• Multiple antibody validation: Use two different At5g35735 antibodies targeting different epitopes

• Correlation with mRNA expression: Compare protein detection with transcript levels from RNA-seq or qPCR

Document validation results thoroughly to establish confidence in experimental findings and address potential reviewer concerns.

How can At5g35735 antibodies be integrated with high-throughput experimental approaches?

Modern high-throughput experiments can incorporate At5g35735 antibodies in several ways:

• Protein microarrays: For analyzing interactions with other proteins or potential ligands

• Single-cell protein analysis: Combining with flow cytometry for cell-specific expression patterns

• Temporal proteomics: Tracking At5g35735 dynamics during infection progression

• Multi-omics integration: Correlating antibody-detected protein levels with transcriptomics and metabolomics data

These approaches align with integrated technology pipelines similar to those used for accelerated discovery of antiviral antibodies , providing comprehensive insights into At5g35735 function in complex biological contexts.

What are common challenges in At5g35735 antibody experiments and how can they be addressed?

Researchers frequently encounter these challenges when working with plant antibodies like At5g35735:

When troubleshooting, systematically modify one variable at a time and document all changes to experimental conditions.

How should researchers interpret contradictory results between antibody detection and transcript analysis?

Discrepancies between protein and mRNA levels are common in biological systems and may reveal important regulatory mechanisms:

• Post-transcriptional regulation: Investigate microRNA targeting, RNA binding proteins, or mRNA stability factors

• Translational control: Examine potential regulation at the translation level

• Protein stability: Assess post-translational modifications or proteasomal degradation pathways

• Temporal dynamics: Consider time-lag between transcription and translation

• Technical artifacts: Rule out antibody cross-reactivity or primer specificity issues

These discrepancies may be particularly informative when studying stress responses like pathogen infection, where regulatory networks often involve multiple layers of control .

What considerations are important when analyzing At5g35735 in the context of pathogen response networks?

When analyzing At5g35735 in pathogen response networks, consider these analytical frameworks:

• Network position analysis: Identify whether At5g35735 functions as a hub, bottleneck, or peripheral component in protein interaction networks

• Temporal dynamics: Track expression changes across infection time-course, comparing with known defense response markers

• Comparative analysis: Contrast responses between different pathogen types (e.g., Salmonella WT vs. prgH-)

• Pathway enrichment: Determine which signaling pathways are statistically associated with At5g35735 regulation

• Cross-species comparisons: Compare with homologous proteins in other plant systems to identify conserved defense mechanisms

Integration with computational models such as those used in signal transduction analysis can reveal master regulators and key network components in specific infection contexts .

What are emerging research directions for At5g35735 antibody applications?

Future research using At5g35735 antibodies may explore:

• Single-cell resolution studies: Examining cell-type specific expression during infection

• Structural biology integration: Combining antibody-based detection with structural analyses

• Systems biology approaches: Positioning At5g35735 within larger regulatory networks

• Comparative plant immunity: Analyzing homologs across plant species for evolutionary insights

• Targeted protein engineering: Modifying At5g35735 function based on antibody-derived structural information

These directions align with broader trends in plant immunity research and may yield insights into fundamental biological processes governing plant-pathogen interactions .

How should researchers evaluate new At5g35735 antibodies for their specific applications?

When evaluating new At5g35735 antibodies for specific applications, researchers should:

• Request validation data from manufacturers

• Conduct preliminary tests comparing multiple antibodies under identical conditions

• Validate using knockout/knockdown plants as negative controls

• Optimize protocols specifically for the intended application

• Document batch variation and establish quality control metrics

Remember that antibody performance can vary significantly between applications (Western blotting vs. immunoprecipitation vs. immunofluorescence), necessitating application-specific validation protocols.