At1g77880 Antibody

What is At1g77880 and why is it studied in Arabidopsis research?

At1g77880 (UniProt ID: Q9SH13) is a protein-coding gene in Arabidopsis thaliana (Mouse-ear cress), a model organism extensively used in plant molecular biology research. This protein is studied because understanding its function contributes to our knowledge of plant development, cellular signaling, and response mechanisms. The antibody against this protein serves as an essential tool for detecting, quantifying, and localizing the protein in various experimental contexts. Unlike antibodies against animal proteins, plant-specific antibodies like At1g77880 require specific validation approaches due to the unique challenges of plant cellular components .

What are the optimal storage conditions for maintaining At1g77880 antibody activity?

For optimal preservation of At1g77880 antibody activity, store the antibody at -20°C or -80°C upon receipt. Avoid repeated freeze-thaw cycles, as these can significantly compromise antibody function and specificity. The antibody is supplied in a protective storage buffer containing 0.03% Proclin 300 preservative, 50% Glycerol, and 0.01M PBS at pH 7.4, which helps maintain stability during long-term storage. For experiments requiring repeated use, consider aliquoting the antibody into single-use volumes before freezing to minimize freeze-thaw damage .

What applications has the At1g77880 antibody been validated for?

The At1g77880 antibody has been tested and validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications. These techniques provide different but complementary information: ELISA allows for quantitative measurement of the target protein in solution, while Western Blotting enables visualization of protein expression, molecular weight verification, and semi-quantitative analysis of protein levels. When using this antibody for other applications such as immunohistochemistry or chromatin immunoprecipitation, additional validation steps should be performed to ensure specificity and reliability .

How should researchers prepare samples for optimal At1g77880 antibody detection?

Sample preparation is critical for successful antibody detection. For Arabidopsis thaliana tissues, use a protein extraction buffer that preserves protein integrity while efficiently extracting the target protein. A typical extraction buffer might include 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail. For Western blotting, denature proteins at 95°C for 5 minutes in sample buffer containing SDS and β-mercaptoethanol. When working with plant tissues, additional steps to remove interfering compounds like polyphenols and polysaccharides may be necessary to improve detection specificity and reduce background .

How do post-translational modifications of At1g77880 affect antibody recognition?

Post-translational modifications (PTMs) can significantly impact antibody recognition of At1g77880. The polyclonal nature of this antibody means it recognizes multiple epitopes on the target protein, potentially including regions subject to phosphorylation, glycosylation, or other modifications. When investigating PTMs, consider:

For definitive studies of protein modifications, consider combining this antibody with modification-specific antibodies or mass spectrometry approaches .

What strategies can resolve cross-reactivity issues with the At1g77880 antibody?

Despite the antibody being antigen affinity-purified, cross-reactivity with similar plant proteins remains a potential concern. To address this:

• Pre-absorption controls: Incubate the antibody with purified recombinant At1g77880 protein before immunodetection to verify signal specificity.

• Genetic controls: Compare antibody signal between wild-type plants and At1g77880 knockout/knockdown lines. A specific antibody should show reduced or absent signal in genetic knockouts.

• Epitope competition assay: Perform parallel experiments with and without excess immunizing peptide to compete for antibody binding.

• Orthogonal detection methods: Validate findings using alternative approaches like mass spectrometry or RNA expression analysis.

These approaches help distinguish true At1g77880 signal from potential cross-reactivity with homologous proteins in complex plant extracts .

How can At1g77880 antibody be used to investigate protein-protein interactions?

The At1g77880 antibody can be employed in several experimental approaches to study protein-protein interactions:

• Co-immunoprecipitation (Co-IP): Use the antibody to pull down At1g77880 protein complexes from plant extracts, followed by mass spectrometry or Western blotting to identify interacting partners.

• Proximity labeling: Combine the antibody with techniques like BioID or APEX, where the antibody localizes the enzymatic activity to label proximal proteins.

• Duolink proximity ligation assay: Detect protein interactions in situ by combining At1g77880 antibody with antibodies against potential interacting partners.

• Chromatin immunoprecipitation (ChIP): If At1g77880 functions in transcriptional regulation, ChIP can identify DNA binding sites and co-factors.

When studying F-box proteins like those in Arabidopsis, consider that protein interactions may be transient or condition-dependent, requiring crosslinking approaches or specialized buffer conditions to preserve interactions during experimental manipulation .

What methodological considerations are important when comparing At1g77880 expression across different plant stress conditions?

When investigating At1g77880 expression under various stress conditions, consider these methodological factors:

• Consistent sampling: Harvest tissues at the same developmental stage and time of day to minimize circadian or developmental variation.

• Stress application standardization: Apply stresses consistently across experiments, documenting parameters like duration, intensity, and recovery periods.

• Multiple detection methods: Combine antibody-based protein detection with transcript analysis to distinguish between transcriptional and post-transcriptional regulation.

• Loading controls: Use established housekeeping proteins or total protein staining methods appropriate for the stress condition being tested, as some traditional controls may themselves be stress-responsive.

• Temporal dynamics: Perform time-course experiments to capture both early and late responses, as protein expression patterns may change dynamically during stress adaptation.

Similar approaches have been successful in characterizing stress-responsive proteins like At1g08710, which shows altered expression patterns under drought stress conditions .

What are the optimal dilution ratios and incubation conditions for At1g77880 antibody in different applications?

Determining optimal working conditions for the At1g77880 antibody is essential for generating reliable and reproducible results:

For novel applications, begin with manufacturer's recommendations and adjust based on empirical results. When optimizing, change only one parameter at a time while keeping others constant to systematically determine optimal conditions .

How can researchers validate At1g77880 antibody specificity in Arabidopsis mutant lines?

Antibody validation using genetic approaches is the gold standard for confirming specificity:

• Knockout/knockdown comparison: Compare antibody signal between wild-type plants and At1g77880 knockout or knockdown lines. A specific antibody will show significantly reduced or absent signal in genetic mutants.

• Overexpression analysis: In complementary experiments, examine plants overexpressing At1g77880 to confirm increased signal intensity correlating with expression level.

• Epitope-tagged proteins: Compare detection using the At1g77880 antibody versus an antibody against an epitope tag fused to At1g77880.

• Recombinant protein control: Include purified recombinant At1g77880 protein as a positive control to verify the expected molecular weight and signal characteristics.

This multi-faceted validation approach, similar to that used for F-box proteins like At1g08710, ensures confidence in experimental results and facilitates troubleshooting when unexpected patterns are observed .

What methods can enhance At1g77880 detection sensitivity in samples with low expression levels?

When working with samples where At1g77880 is expressed at low levels, several techniques can enhance detection sensitivity:

• Sample enrichment: Increase protein concentration through immunoprecipitation or subcellular fractionation to enrich for compartments where At1g77880 is localized.

• Signal amplification: Employ tyramide signal amplification (TSA) or polymer-based detection systems that provide higher sensitivity than conventional secondary antibody methods.

• Extended exposure times: For Western blots, use longer exposure times with low-noise detection systems like cooled CCD cameras.

• Alternative visualization: Consider using chemiluminescent substrates with higher sensitivity or fluorescent secondary antibodies with appropriate imaging systems.

• Protein stabilization: If At1g77880 has high turnover, treat samples with proteasome inhibitors before extraction to prevent degradation and increase detectable levels.

These approaches can significantly improve detection of low-abundance plant proteins while maintaining signal specificity .

How does the performance of At1g77880 antibody compare with antibodies for related plant proteins?

Comparative analysis of different plant protein antibodies reveals important considerations for experimental design:

While At1g77880 antibody has been specifically validated for ELISA and Western blot applications, antibodies against more extensively studied plant proteins might offer broader application validation. Consider these differences when designing multi-protein detection experiments or when selecting controls .

What role might At1g77880 play in plant stress responses based on current research?

While specific information about At1g77880's role in stress responses is limited in the provided search results, we can draw parallels with related Arabidopsis proteins:

F-box proteins in Arabidopsis, like At1g08710, have been implicated in drought stress adaptation through modulation of reactive oxygen species and interaction with transcriptional regulators. These proteins often function within SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase complexes to regulate protein degradation in response to environmental stimuli.

To investigate At1g77880's potential role in stress responses:

• Analyze promoter elements for stress-responsive motifs

• Monitor protein expression changes under various stress conditions

• Examine phenotypes of At1g77880 mutant plants under stress

• Identify protein interaction partners that may link to stress signaling pathways

Such approaches have successfully elucidated the functions of other Arabidopsis proteins in stress adaptation mechanisms .

What are the key considerations when designing immunohistochemistry experiments with At1g77880 antibody?

Although the At1g77880 antibody hasn't been specifically validated for immunohistochemistry (IHC), researchers planning such experiments should consider:

• Fixation optimization: Test multiple fixatives (e.g., paraformaldehyde, glutaraldehyde) and fixation times to preserve epitope accessibility while maintaining tissue morphology.

• Antigen retrieval: Evaluate heat-induced or enzymatic antigen retrieval methods to expose epitopes that may be masked during fixation.

• Plant-specific challenges: Address autofluorescence from chlorophyll and cell wall components using appropriate quenching techniques or spectral unmixing during imaging.

• Controls: Include negative controls (secondary antibody only, pre-immune serum) and biological controls (tissues known to express or lack At1g77880) in each experiment.

• Signal validation: Confirm specificity by comparing immunofluorescence patterns with other localization methods like fluorescent protein fusions.

These considerations help overcome the unique challenges posed by plant tissues in immunohistochemistry applications .

How might new antibody technologies enhance At1g77880 research?

Emerging antibody technologies present exciting opportunities for advancing At1g77880 research:

• Recombinant antibody fragments: Single-chain variable fragments (scFvs) or nanobodies derived from conventional antibodies offer improved tissue penetration and production consistency.

• Bifunctional antibodies: Engineered antibodies that simultaneously bind At1g77880 and a second target could enable novel approaches to studying protein interactions or localization.

• Conditionally stable antibody fragments: Destabilized antibody domains that become stable only upon target binding provide opportunities for real-time monitoring of protein dynamics.

• CRISPR-based epitope tagging: Precise genomic integration of tags at the endogenous At1g77880 locus enables antibody detection while maintaining native regulation.

These technologies could overcome current limitations in studying low-abundance or conditionally expressed plant proteins under physiologically relevant conditions .

What methodological approaches can integrate At1g77880 antibody data with other omics datasets?

Integrating antibody-based protein detection with multi-omics data provides a comprehensive understanding of At1g77880 function:

• Proteogenomics: Correlate At1g77880 protein levels with transcript abundance data to identify post-transcriptional regulation mechanisms.

• Protein interaction networks: Combine immunoprecipitation results with predicted interaction networks from genomic and transcriptomic data.

• Functional genomics integration: Compare phenotypic data from At1g77880 mutants with protein expression patterns to establish structure-function relationships.

• Systems biology modeling: Incorporate quantitative antibody-based measurements into mathematical models of plant response pathways.

• Metadata standardization: Ensure experimental conditions and antibody validation parameters are comprehensively documented to facilitate meta-analysis across studies.

This integrative approach has proven valuable in characterizing the functional roles of other Arabidopsis proteins like the F-box protein At1g08710 in drought stress adaptation .