At5g24760 Antibody

What is the At5g24760 protein and why is it studied in plant research?

At5g24760 (UniProt Number: Q8LEB2) is a protein found in Arabidopsis thaliana with significance in plant molecular biology research. The protein is studied to understand its specific functions in plant cellular processes, including potential roles in stress response mechanisms and developmental pathways. Understanding this protein contributes to our broader knowledge of plant biology and potentially agricultural applications. Research involving At5g24760 typically employs antibody-based detection methods to identify expression patterns, localization, and protein-protein interactions .

What are the key specifications of the At5g24760 antibody?

The At5g24760 antibody is a rabbit polyclonal antibody generated against recombinant Arabidopsis thaliana At5g24760 protein. It is supplied as an affinity-purified antibody with IgG isotype. The antibody is unconjugated (not linked to any reporter molecules) and designed for plant species reactivity. Standard commercial preparations include 0.2mg of purified antibody along with 200μg of antigen (for positive control) and 1ml pre-immune serum (for negative control). The antibody has been validated for use in ELISA and Western blot applications .

How should the At5g24760 antibody be stored and handled to maintain optimal activity?

For optimal preservation of antibody function, store the At5g24760 antibody at -20°C or -80°C in small aliquots to minimize freeze-thaw cycles. Similar to other research antibodies, each freeze-thaw cycle can reduce activity by approximately 10%, with significant degradation occurring after 5+ cycles. When handling the antibody:

• Thaw aliquots on ice rather than at room temperature

• Centrifuge briefly before opening tubes to collect all liquid

• Use sterile technique when accessing stock solutions

• Return to -20°C or -80°C immediately after use

This storage protocol follows standard practices for polyclonal antibodies targeting plant proteins .

What is the recommended protocol for Western blot analysis using At5g24760 antibody?

For optimal Western blot results with At5g24760 antibody, follow this methodological approach:

Sample Preparation:

• Extract plant proteins using a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, and protease inhibitors

• Quantify protein concentration using Bradford or BCA assay

• Denature 20-50μg protein in Laemmli buffer (with DTT or β-mercaptoethanol) at 95°C for 5 minutes

Gel Electrophoresis and Transfer:

• Separate proteins on 10-12% SDS-PAGE

• Transfer to PVDF membrane (0.45μm) using semi-dry or wet transfer

• Verify transfer efficiency with Ponceau S staining

Immunodetection:

• Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature

• Incubate with At5g24760 antibody at 1:1000 dilution in blocking buffer overnight at 4°C

• Wash 3× with TBST, 10 minutes each

• Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour

• Wash 3× with TBST, 10 minutes each

• Develop using ECL substrate and document results

Critical Controls:

• Include pre-immune serum at the same dilution as primary antibody (negative control)

• Include purified antigen or overexpression lysate (positive control)

• Include molecular weight markers to verify target protein size

This protocol is based on standard western blotting procedures for plant proteins and should be optimized based on specific research conditions .

How should I design experiments to validate At5g24760 antibody specificity?

Validation of At5g24760 antibody specificity requires a multi-faceted approach to eliminate false positives and confirm target recognition:

Primary Validation Methods:

• Peptide Competition Assay:

  • Pre-incubate antibody with excess purified At5g24760 antigen (supplied with antibody)

  • Run parallel Western blots with neutralized and non-neutralized antibody

  • Specific signals should be absent in the neutralized antibody lane

• Genetic Controls:

  • Test antibody against wild-type and knockout/knockdown plant tissues

  • Specific signal should be absent or reduced in knockout/knockdown samples

  • Alternatively, test against tissue with At5g24760 overexpression

• Mass Spectrometry Validation:

  • Perform immunoprecipitation with the At5g24760 antibody

  • Analyze pulled-down proteins by LC-MS/MS

  • Confirm presence of At5g24760 in the immunoprecipitated fraction

Validation Data Presentation:

This comprehensive validation strategy ensures that experimental results using the antibody accurately reflect the biological presence and behavior of the At5g24760 protein .

What are the optimal conditions for ELISA using At5g24760 antibody?

For developing a robust ELISA protocol with At5g24760 antibody, the following methodological approach is recommended:

Indirect ELISA Protocol:

• Plate Coating:

  • Dilute protein samples in carbonate/bicarbonate buffer (pH 9.6)

  • Coat 96-well plates with 100μl sample per well

  • Incubate overnight at 4°C

• Blocking and Primary Antibody:

  • Wash 3× with PBST (PBS + 0.05% Tween-20)

  • Block with 300μl 3% BSA in PBS for 1 hour at room temperature

  • Add At5g24760 antibody diluted 1:2000 in 1% BSA/PBS

  • Incubate for 2 hours at room temperature

• Detection:

  • Wash 4× with PBST

  • Add HRP-conjugated anti-rabbit secondary antibody (1:5000)

  • Incubate for 1 hour at room temperature

  • Wash 4× with PBST

  • Add 100μl TMB substrate

  • Stop reaction with 100μl 2N H₂SO₄ after 15-30 minutes

  • Read absorbance at 450nm

Sandwich ELISA Alternative:
For higher specificity, a sandwich ELISA can be developed using a capture antibody against a different epitope of At5g24760, then using the affinity-purified antibody as a detection antibody.

Optimization Parameters:

Always include a standard curve using the provided antigen (200μg) and the pre-immune serum as a negative control to establish assay specificity and sensitivity .

What are common issues in Western blot analysis with At5g24760 antibody and how can they be resolved?

When working with At5g24760 antibody in Western blot applications, researchers may encounter several technical challenges. This comprehensive troubleshooting guide addresses common issues:

No Signal:

• Potential Causes:

  • Insufficient protein loading

  • Protein degradation

  • Inefficient transfer

  • Antibody denaturation

  • Incorrect primary or secondary antibody dilution

• Solutions:

  • Increase protein loading to 40-50μg

  • Add fresh protease inhibitors to extraction buffer

  • Verify transfer with Ponceau S staining

  • Use fresh antibody aliquot

  • Optimize antibody dilution (try 1:500 instead of 1:1000)

  • Extend primary antibody incubation to overnight at 4°C

Multiple Bands:

• Potential Causes:

  • Protein degradation

  • Cross-reactivity with related proteins

  • Post-translational modifications

  • Non-specific binding

• Solutions:

  • Use freshly prepared samples with protease inhibitors

  • Increase blocking time and concentration (5% to 7% BSA)

  • Reduce primary antibody concentration (1:2000 instead of 1:1000)

  • Add 0.1% SDS to antibody dilution buffer to reduce non-specific binding

  • Run peptide competition assay to identify specific band

High Background:

• Potential Causes:

  • Insufficient blocking

  • Too concentrated antibody

  • Insufficient washing

  • Membrane overexposure

• Solutions:

  • Extend blocking time to 2 hours

  • Increase blocking agent concentration to 5%

  • Dilute primary antibody further (1:2000)

  • Add 0.05-0.1% Tween-20 to washing buffer

  • Perform additional or longer washing steps (5× 10 minutes)

  • Reduce exposure time during imaging

This methodological troubleshooting approach should help resolve common issues encountered when using the At5g24760 antibody in Western blot applications .

How can I interpret contradictory results between Western blot and ELISA when using At5g24760 antibody?

When faced with discrepancies between Western blot and ELISA results using the At5g24760 antibody, a systematic analytical approach is essential:

Understanding Fundamental Differences:

Analytical Framework for Resolving Contradictions:

• Positive in ELISA, Negative in Western:

  • The antibody may recognize conformational epitopes disrupted by denaturation

  • Solution: Try non-denaturing conditions in Western or dot blot

  • Methodological approach: Run native PAGE or reduce SDS concentration

• Positive in Western, Negative in ELISA:

  • The epitope may be masked in the native state

  • Solution: Try different coating buffers or mild denaturation for ELISA

  • Methodological approach: Test pH range 7.0-9.6 for coating or add 0.5-1% SDS

• Different Quantitative Results:

  • Different sensitivities between techniques

  • Matrix effects in complex samples

  • Solution: Prepare standard curves in matched matrices

  • Methodological approach: Spike known quantities of antigen into sample matrix

• Experimental Validation Strategy:

  • Run parallel experiments with positive controls

  • Test different sample preparation methods

  • Consider protein extraction protocols that maintain native state for ELISA

  • Use recombinant At5g24760 protein to calibrate both assays

When interpreting contradictory results, remember that Western blot provides information about protein size and specificity, while ELISA offers quantitative measurement with potentially higher sensitivity. The techniques should be viewed as complementary rather than contradictory .

What controls are essential when working with At5g24760 antibody to ensure result validity?

Robust experimental design with appropriate controls is critical for generating reliable data with the At5g24760 antibody. The following comprehensive control strategy should be implemented:

Essential Controls for All Experiments:

• Negative Controls:

  • Pre-immune serum (supplied with antibody): Apply at the same dilution as primary antibody

  • Secondary antibody only: Omit primary antibody to detect non-specific binding

  • Isotype control: Use non-specific rabbit IgG at equivalent concentration

• Positive Controls:

  • Purified antigen (supplied with antibody): Use the 200μg provided antigen

  • Overexpression system: Arabidopsis plants or protoplasts overexpressing At5g24760

  • Known positive tissue: Tissues with verified high expression of At5g24760

• Genetic Controls:

  • Wild-type vs. knockout/knockdown: Compare signals between plants with and without At5g24760 expression

  • Dosage response: Compare heterozygous vs. homozygous mutants or overexpression lines

Application-Specific Controls:

Data Validation Framework:

• Each experiment should include at minimum: one negative control, one positive control, and appropriate loading/technical controls

• Results should be reproducible across at least three biological replicates

• Control responses should be consistent across experiments

This comprehensive control strategy ensures that results obtained with the At5g24760 antibody can be confidently interpreted as reflecting genuine biological phenomena rather than technical artifacts .

How can the At5g24760 antibody be used for co-immunoprecipitation studies to identify protein interaction partners?

Co-immunoprecipitation (Co-IP) is a powerful approach for identifying protein-protein interactions involving At5g24760. This methodological protocol outlines the complete workflow:

Sample Preparation:

• Harvest 5-10g plant tissue and flash-freeze in liquid nitrogen

• Grind tissue to fine powder while maintaining freezing conditions

• Extract proteins in non-denaturing buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 0.5% NP-40, 1mM EDTA, protease inhibitors)

• Clarify lysate by centrifugation (14,000×g, 15 minutes, 4°C)

• Pre-clear with Protein A beads (1 hour, 4°C with rotation)

Immunoprecipitation:

• Add At5g24760 antibody (5-10μg) to 1ml pre-cleared lysate

• Incubate overnight at 4°C with gentle rotation

• Add 50μl Protein A magnetic beads

• Incubate 3 hours at 4°C with gentle rotation

• Collect beads using magnetic stand

• Wash 5× with IP buffer (reduced detergent - 0.1% NP-40)

• Elute proteins with 2× Laemmli buffer or native elution buffer

Analysis Options:

• SDS-PAGE/Western Blot:

  • Run samples on 10-12% gel

  • Transfer to membrane

  • Probe with antibodies against suspected interaction partners

• Mass Spectrometry:

  • Run samples on short SDS-PAGE (minimal separation)

  • Excise gel sections

  • Process for in-gel digestion with trypsin

  • Analyze peptides by LC-MS/MS

  • Compare to control IP (pre-immune serum) to identify specific interactors

Validation Strategy:

• Confirm key interactions with reciprocal Co-IP

• Verify with alternative methods (Y2H, BiFC, FRET)

• Test interaction dependency on experimental conditions (salt concentration, pH)

Interaction Network Analysis:
Create a data table of identified partners with these parameters:

This comprehensive Co-IP approach enables researchers to uncover the interactome of At5g24760, providing insights into its biological functions through protein interaction networks .

What are the considerations for using At5g24760 antibody in chromatin immunoprecipitation (ChIP) studies?

While the At5g24760 antibody is primarily validated for ELISA and Western blot applications, researchers may consider adapting it for chromatin immunoprecipitation studies if At5g24760 is suspected to interact with DNA or chromatin-associated complexes. This methodological guide outlines special considerations:

Antibody Suitability Assessment:

• Epitope Accessibility:

  • Determine if the epitope remains accessible when At5g24760 is bound to chromatin

  • Test antibody in nuclear fractionation Western blot before proceeding

  • Verify nuclear localization via immunofluorescence

• Crosslinking Compatibility:

  • Some epitopes may be masked by formaldehyde crosslinking

  • Consider testing both crosslinked and native ChIP protocols

  • Begin with lower formaldehyde concentrations (0.75% instead of 1%)

Optimized ChIP Protocol:

• Chromatin Preparation:

  • Crosslink tissue with 1% formaldehyde for 10 minutes

  • Quench with 125mM glycine

  • Isolate nuclei and sonicate to generate 200-500bp fragments

  • Verify fragmentation by agarose gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with Protein A beads

  • Incubate 5-10μg At5g24760 antibody with chromatin overnight

  • Include parallel IPs with pre-immune serum and no-antibody controls

  • Wash stringently to remove non-specific interactions

  • Reverse crosslinks and purify DNA

• Analysis Options:

  • ChIP-qPCR for targeted loci

  • ChIP-seq for genome-wide binding profile

Validation Requirements:

Troubleshooting ChIP-Specific Issues:

• Low Enrichment:

  • Increase antibody amount (10-15μg)

  • Reduce chromatin amount

  • Optimize crosslinking time and sonication

  • Try different epitope exposure methods

• High Background:

  • Increase wash stringency (higher salt concentration)

  • Extend washing time

  • Add competitor DNA/protein to reduce non-specific binding

This advanced application requires careful optimization and validation, as the At5g24760 antibody was not specifically validated for ChIP applications in the original product specifications .

How can At5g24760 antibody be integrated with advanced proteomics techniques for plant stress response studies?

Integrating At5g24760 antibody with cutting-edge proteomics approaches can yield valuable insights into plant stress response mechanisms. This methodological framework outlines advanced applications:

Quantitative Proteomics Workflows:

• Immunoaffinity Purification-Mass Spectrometry (IP-MS):

  • Perform IP with At5g24760 antibody under different stress conditions

  • Process samples for LC-MS/MS analysis

  • Quantify changes in interaction partners using label-free or labeled methods

  • Integrate with phosphoproteomics to identify stress-induced modifications

  Experimental Design:

  Condition	Biological Replicates	Technical Replicates	Controls
  Control	3	2	Pre-immune serum IP
  Drought	3	2	Pre-immune serum IP
  Salt stress	3	2	Pre-immune serum IP
  Cold stress	3	2	Pre-immune serum IP

• Selected Reaction Monitoring (SRM)/Parallel Reaction Monitoring (PRM):

  • Develop targeted MS assays for At5g24760 and related proteins

  • Quantify precise protein levels across stress conditions

  • Monitor specific post-translational modifications

  Implementation Approach:

  • Identify unique peptides for At5g24760 using discovery proteomics

  • Select 3-5 representative peptides spanning the protein sequence

  • Develop SRM/PRM assays with appropriate internal standards

  • Apply to time-course experiments during stress response

• Spatial Proteomics Integration:

  • Combine subcellular fractionation with At5g24760 immunoblotting

  • Track protein relocalization during stress response

  • Correlate with interaction partner dynamics

  Spatial Distribution Analysis:

  Subcellular Fraction	Control Conditions	Stress Conditions	Change
  Cytosolic	[Signal intensity]	[Signal intensity]	[%]
  Nuclear	[Signal intensity]	[Signal intensity]	[%]
  Membrane	[Signal intensity]	[Signal intensity]	[%]
  Organellar	[Signal intensity]	[Signal intensity]	[%]

Systems Biology Integration Framework:

• Multi-omics Data Integration:

  • Correlate At5g24760 protein levels (detected by antibody) with:

    • Transcriptome data (RNA-seq)

    • Metabolome profiles

    • Physiological measurements

• Protein Interaction Network Modeling:

  • Map At5g24760 interaction partners identified by IP-MS

  • Integrate with public interaction databases

  • Identify stress-responsive network modules

  • Predict functional consequences using network analysis

• Validation Through Genetic Approaches:

  • Generate transgenic lines with tagged At5g24760 for complementary analysis

  • Compare antibody-based detection with tag-based detection

  • Correlate protein dynamics with phenotypic responses to stress

This comprehensive integration of the At5g24760 antibody with advanced proteomics techniques provides a powerful approach for understanding the role of this protein in plant stress responses, potentially uncovering novel regulatory mechanisms and stress adaptation pathways .

How does the specificity of At5g24760 antibody compare across different plant species?

When applying the At5g24760 antibody across different plant species, researchers must consider cross-reactivity patterns and conservation of the target protein. This comparative analysis provides methodological guidance:

Cross-Species Reactivity Analysis:

While the At5g24760 antibody was raised against Arabidopsis thaliana protein, potential cross-reactivity with orthologous proteins in other plant species should be systematically evaluated before experimental application. Protein sequence conservation varies significantly across plant lineages.

Methodological Approach for Cross-Species Validation:

• Sequence Homology Assessment:

  • Identify At5g24760 orthologs using BLAST/OrthoFinder

  • Align sequences to determine epitope conservation

  • Predict cross-reactivity based on sequence identity percentage

• Empirical Validation Strategy:

  • Test antibody against protein extracts from multiple species

  • Run Western blots with equal protein loading

  • Compare signal intensity and band specificity

  Recommended Testing Protocol:

  • Extract proteins using identical protocols across species

  • Load 30μg total protein per lane

  • Run alongside Arabidopsis positive control

  • Include molecular weight markers to identify size shifts

Expected Cross-Reactivity Pattern:

Optimization for Cross-Species Applications:

• For High Homology Species:

  • Standard antibody dilution (1:1000-1:2000) may be sufficient

  • Verify single band of expected molecular weight

• For Moderate Homology Species:

  • Reduce antibody dilution (1:500)

  • Increase blocking stringency (5% BSA)

  • Optimize incubation conditions (overnight, 4°C)

• For Low Homology Species:

  • Consider dot blot before Western blot for initial screening

  • May require signal enhancement techniques

  • Validate with genetic controls (e.g., RNAi lines) if available

This systematic approach to cross-species reactivity assessment ensures appropriate application of the At5g24760 antibody across plant species, preventing misinterpretation of results when working with non-Arabidopsis systems .

How can At5g24760 antibody be used to study protein expression changes during plant development stages?

The At5g24760 antibody can be leveraged to investigate developmental regulation of this protein through a systematic temporal and spatial analysis. This methodological framework outlines the approach:

Developmental Expression Analysis Protocol:

• Temporal Profiling:

  • Collect plant material at defined developmental stages

  • Sample key timepoints: seed germination, seedling, vegetative growth, flowering, silique development, senescence

  • Process samples consistently for protein extraction

  • Quantify relative protein levels by Western blot

  Standardized Sampling Framework:

  Developmental Stage	Days After Germination	Tissue Types to Sample
  Germination	0-2	Whole seedling
  Early seedling	3-7	Cotyledon, hypocotyl, root
  Vegetative growth	14-21	Young leaves, mature leaves, root
  Transition to flowering	21-28	Apical meristem, young leaves, stem
  Reproductive	35-42	Flowers (stages 1-12), siliques, stem
  Senescence	49+	Senescing leaves, mature siliques

• Tissue-Specific Expression:

  • Dissect distinct tissues at each developmental stage

  • Extract proteins using optimized buffers for each tissue type

  • Normalize loading by total protein or housekeeping proteins

  • Compare expression patterns across tissues

  Protein Extraction Optimization:

  Tissue Type	Buffer Modifications	Special Considerations
  Leaf	Standard	High RuBisCO content may interfere
  Root	Add 1% PVP	High phenolic content
  Flower	Add 0.5% Triton X-100	Complex tissues require thorough extraction
  Silique	Increase DTT to 5mM	Higher proteolytic activity

• Quantitative Analysis Approach:

  • Use digital imaging and densitometry software

  • Normalize signals to internal controls

  • Plot expression changes over developmental timeline

  • Analyze statistical significance across replicates

Advanced Developmental Analysis Techniques:

• Immunohistochemistry Integration:

  • Fix tissue samples in 4% paraformaldehyde

  • Section tissues (10-20μm)

  • Perform antigen retrieval if necessary

  • Incubate with At5g24760 antibody (1:200)

  • Visualize with fluorescent secondary antibody

  • Counterstain with DAPI for nuclear visualization

• Protein Turnover Analysis:

  • Treat plants with cycloheximide to inhibit protein synthesis

  • Sample tissues at timepoints (0, 3, 6, 12, 24 hours)

  • Analyze At5g24760 protein levels by Western blot

  • Calculate protein half-life across developmental stages

• Environmental Response Integration:

  • Assess how developmental expression patterns change under:

    • Different light conditions

    • Temperature variations

    • Nutrient availability

    • Stress treatments

This comprehensive developmental analysis using the At5g24760 antibody can reveal important insights into protein function throughout the plant life cycle, potentially identifying critical developmental stages where the protein plays key regulatory roles .

What data analysis methods should be used when comparing At5g24760 antibody detection results across multiple experimental conditions?

Robust data analysis is essential when using At5g24760 antibody across multiple experimental conditions. This methodological framework ensures statistical rigor and biological relevance:

Quantitative Data Analysis Workflow:

• Image Acquisition and Densitometry:

  • Capture Western blot images in linear dynamic range

  • Use calibrated imaging systems (ChemiDoc, LI-COR, etc.)

  • Perform densitometry using ImageJ or specialized software

  • Include standards for absolute quantification if needed

• Data Normalization Strategies:

  Normalization Method	Application	Advantages	Limitations
  Housekeeping proteins	Western blot	Well-established	May vary under some conditions
  Total protein (Ponceau)	Western blot/dot blot	Independent of reference proteins	Requires additional steps
  Recombinant protein standards	Western blot/ELISA	Absolute quantification	Requires purified protein
  Sample-to-sample normalization	LC-MS/MS	Accounts for loading variation	Complex implementation

• Statistical Analysis Framework:

  • Perform minimum of 3-4 biological replicates

  • Test data for normality (Shapiro-Wilk test)

  • Apply appropriate statistical tests:

    • For normal data: t-test (two conditions) or ANOVA (multiple conditions)

    • For non-normal data: Mann-Whitney U (two conditions) or Kruskal-Wallis (multiple conditions)

  • Apply post-hoc tests for multiple comparisons (Tukey's HSD, Bonferroni)

  • Calculate effect sizes in addition to p-values

• Data Visualization Best Practices:

  • Present individual data points alongside means

  • Include error bars (standard deviation or standard error)

  • Use consistent scaling for comparative analyses

  • Consider heatmaps for multi-conditional experiments

Advanced Analytical Approaches:

• Multivariate Analysis:

  • Principal Component Analysis (PCA) to identify patterns across conditions

  • Hierarchical clustering to group similar responses

  • Correlation analyses with other measured parameters

• Temporal Data Analysis:

  • Time-series analysis for dynamic responses

  • Area-under-curve calculations for cumulative effects

  • Rate-of-change analysis for response kinetics

• Integration with Transcriptomic Data:

  • Calculate protein-to-mRNA ratios

  • Identify post-transcriptional regulation

  • Analyze time-delays between transcription and translation

Methodological Guidelines for Complex Experimental Designs:

• Factorial Designs:

  • Analyze main effects and interactions

  • Use two-way or three-way ANOVA

  • Calculate interaction terms and significance

• Dose-Response Studies:

  • Fit appropriate models (linear, sigmoidal, etc.)

  • Calculate EC50/IC50 values if applicable

  • Compare curve parameters across conditions

• Meta-Analysis Across Experiments:

  • Standardize data for cross-experimental comparison

  • Apply random-effects models to account for inter-experimental variation

  • Calculate confidence intervals for effect sizes

This comprehensive data analysis framework ensures that researchers can extract maximum biological insight from experiments using the At5g24760 antibody across multiple conditions, with appropriate statistical rigor and analytical depth .

What are emerging applications for At5g24760 antibody in plant systems biology?

The At5g24760 antibody offers exciting potential beyond traditional applications, particularly in emerging systems biology approaches. This forward-looking analysis outlines methodological innovations:

Emerging Research Applications:

• Single-Cell Proteomics Integration:

  • Adaptation for plant protoplast flow cytometry

  • Cell type-specific expression profiling

  • Correlation with single-cell transcriptomics

  • Methodological approach: Optimize fixation and permeabilization protocols for maintaining cellular integrity while allowing antibody penetration

• Spatial Proteomics Approaches:

  • In situ immunolocalization with super-resolution microscopy

  • Subcellular fractionation coupled with antibody detection

  • Proximity labeling combined with immunoprecipitation

  • Methodological approach: Combine with genetically encoded markers for co-localization studies and protein proximity analysis

• Stress Granule and Protein Condensate Studies:

  • Investigate At5g24760 association with biomolecular condensates

  • Monitor stress-induced relocalization

  • Study protein-protein interactions within condensates

  • Methodological approach: Implement fluorescence recovery after photobleaching (FRAP) analyses with immunofluorescence

Integration with Advanced Technologies:

Future Research Direction Framework:

• Climate Change Response Studies:

  • Monitor At5g24760 expression under extreme weather conditions

  • Analyze protein modifications in response to combined stresses

  • Correlate with plant adaptation mechanisms

  • Methodological approach: Design multi-factorial experiments with proper controls for individual and combined stress factors

• Crop Improvement Applications:

  • Comparative analysis across crop varieties with varying stress tolerance

  • Investigation of At5g24760 orthologs in major crops

  • Correlation of protein levels with agronomic traits

  • Methodological approach: Develop standardized protocols for field-to-lab sample collection and preservation

• Interactome Mapping:

  • Comprehensive identification of interaction partners across conditions

  • Construction of regulatory networks involving At5g24760

  • Temporal dynamics of protein complexes

  • Methodological approach: Implement BioID or TurboID proximity labeling with At5g24760 as bait protein

This forward-looking perspective highlights how the At5g24760 antibody can be integrated into cutting-edge research paradigms, contributing to our understanding of plant biology in the context of changing environments and advancing agricultural innovation .

What are the most significant knowledge gaps that could be addressed using At5g24760 antibody in plant research?

Strategic application of the At5g24760 antibody can address several critical knowledge gaps in plant molecular biology. This research roadmap outlines key areas for investigation:

Critical Knowledge Gaps and Research Strategies:

• Protein Function and Regulation:

  • Current gap: Limited understanding of At5g24760's precise molecular function

  • Research approach: Combine antibody-based detection with activity assays to correlate protein levels with functional outputs

  • Methodological innovation: Develop co-immunoprecipitation coupled with activity measurements to assess functional protein complexes

• Post-Translational Modifications:

  • Current gap: Unknown regulatory modifications affecting At5g24760

  • Research approach: Immunoprecipitate At5g24760 followed by mass spectrometry to identify phosphorylation, ubiquitination, and other modifications

  • Methodological innovation: Compare modification patterns across developmental stages and stress conditions

  PTM Analysis Framework:

  Modification Type	Detection Method	Functional Validation Approach
  Phosphorylation	Phospho-specific antibodies or MS	Phosphomimetic mutations
  Ubiquitination	IP-MS or ubiquitin-specific antibodies	Proteasome inhibition studies
  SUMOylation	IP-MS or SUMO-specific antibodies	SUMO site mutations
  Redox modifications	Redox-sensitive staining or MS	Oxidative stress treatments

• Tissue-Specific Regulatory Networks:

  • Current gap: Incomplete understanding of tissue-specific functions

  • Research approach: Tissue-specific expression profiling combined with co-expression analysis

  • Methodological innovation: Single-cell resolution immunofluorescence correlated with transcriptomics

• Evolutionary Conservation and Divergence:

  • Current gap: Limited cross-species functional comparison

  • Research approach: Comparative analysis of orthologous proteins across plant lineages

  • Methodological innovation: Develop standardized cross-species analytical protocols

Integrated Research Framework:

• From Correlation to Causation:

  • Current gap: Associative data without causal mechanisms

  • Research approach: Combine antibody detection with genetic manipulation

  • Methodological innovation: Time-resolved analysis of protein dynamics following inducible gene expression/suppression

• Environmental Response Integration:

  • Current gap: Fragmented understanding of environmental response mechanisms

  • Research approach: Multi-stress analysis with protein-level resolution

  • Methodological innovation: Design factorial experiments with comprehensive controls

  Environmental Response Matrix:

  Environmental Factor	Temporal Resolution	Spatial Resolution	Integration with Physiology
  Drought	Short-term vs. chronic	Tissue-specific responses	Water potential correlation
  Temperature extremes	Immediate vs. acclimation	Organ-specific patterns	Metabolic adjustments
  Pathogen exposure	Early vs. late response	Cell-type specificity	Defense marker correlation
  Nutrient availability	Deficiency progression	Root vs. shoot signaling	Growth parameter correlation

• Translational Research Potential:

  • Current gap: Limited application to crop improvement

  • Research approach: Validate findings in crop orthologs

  • Methodological innovation: Develop high-throughput screening protocols

By systematically addressing these knowledge gaps using the At5g24760 antibody as a research tool, scientists can significantly advance our understanding of plant molecular biology, potentially contributing to improved crop resilience and productivity in the face of climate change and food security challenges .

What methodological advances would improve At5g24760 antibody applications in plant research?

To maximize the utility of the At5g24760 antibody in advancing plant research, several methodological innovations should be considered. This forward-looking analysis outlines key technical developments:

Antibody Technology Enhancements:

• Fragment-Based Applications:

  • Development of Fab or scFv derivatives for improved tissue penetration

  • Application in super-resolution microscopy requiring smaller probes

  • Methodological approach: Enzymatic digestion or recombinant production of antibody fragments with retained specificity

• Conjugation Innovations:

  • Direct fluorophore conjugation for one-step immunofluorescence

  • Enzyme-coupled variants for enhanced detection sensitivity

  • Biotin/streptavidin systems for signal amplification

  • Methodological approach: Optimize conjugation chemistry to preserve epitope recognition

• Epitope-Specific Variants:

  • Development of antibodies against different regions of At5g24760

  • Phospho-specific antibodies for regulatory site monitoring

  • Conformation-specific antibodies to detect structural changes

  • Methodological approach: Design strategic immunogens for targeted antibody production

Protocol Optimization Framework:

Emerging Methodological Platforms:

• Microfluidic Integration:

  • Single-cell Western blot applications

  • Automated immunoassay miniaturization

  • Real-time monitoring of protein dynamics

  • Methodological approach: Adapt traditional protocols to microfluidic platforms with reduced sample requirements

• Advanced Microscopy Techniques:

  • Expansion microscopy for improved subcellular resolution

  • Light-sheet microscopy for whole-tissue imaging

  • Correlative light-electron microscopy for ultrastructural context

  • Methodological approach: Optimize fixation and antibody incubation for compatibility with advanced imaging

• Proteomics Interface Development:

  • Antibody-guided mass spectrometry

  • Immunoprecipitation workflows for targeted proteomics

  • Cross-linking mass spectrometry for interaction partner identification

  • Methodological approach: Develop integrated workflows connecting immunocapture with mass spectrometry analysis

Accessibility and Standardization:

• Open Science Resources:

  • Detailed protocol repositories with troubleshooting guides

  • Standard operating procedures for cross-laboratory reproducibility

  • Validation dataset sharing for quality assessment

  • Methodological approach: Establish community standards for antibody validation and application

• Cross-Platform Validation:

  • Correlation between antibody-based and orthogonal detection methods

  • Integration with genomic editing validation

  • Standardized positive and negative controls

  • Methodological approach: Design experiment templates that incorporate multiple detection technologies