At1g31080 Antibody

What is At1g31080 protein and what is its function in Arabidopsis thaliana?

At1g31080 (UniProt ID: Q9SA02) is a protein found in Arabidopsis thaliana (Mouse-ear cress). While the specific research literature on this protein is limited in the provided sources, it belongs to a family of proteins expressed in Arabidopsis that are important for plant development and stress responses. Methodologically, researchers investigating this protein typically employ a combination of genetic approaches (such as knockout/knockdown studies) and protein localization techniques to elucidate its function .

What are the key specifications of commercially available At1g31080 antibodies?

At1g31080 antibodies are typically polyclonal antibodies raised in rabbits against recombinant Arabidopsis thaliana At1g31080 protein. They are generally supplied in liquid form, preserved in a buffer containing approximately 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative. These antibodies are purified using antigen affinity methods and are specifically reactive with Arabidopsis thaliana samples. They are validated for ELISA and Western blot applications to ensure proper identification of the target antigen .

What is the expected molecular weight of At1g31080 protein in Western blot analyses?

While the exact molecular weight is not specified in the provided search results for At1g31080 specifically, researchers should first consult the UniProt database (Q9SA02) to determine the predicted molecular weight based on amino acid sequence. Similar plant proteins, such as actin, typically show differences between predicted molecular weight and apparent molecular weight on SDS-PAGE. For example, actin has a predicted molecular weight of 41.6 kDa but typically appears at approximately 45 kDa on gels . This difference could be due to post-translational modifications or structural features affecting migration patterns.

What are the optimal sample preparation protocols for At1g31080 antibody in Western blotting?

For optimal Western blot results with plant protein antibodies similar to At1g31080:

• Tissue extraction: Harvest fresh Arabidopsis thaliana tissue and immediately freeze in liquid nitrogen. Grind to a fine powder while maintaining frozen state.

• Protein extraction buffer: Use a buffer containing:

  • 0.1 M Tris-HCl (pH 8.0)

  • 5% sucrose (m/v)

  • 2% SDS (m/v)

  • Protease inhibitors (1 mM PMSF or commercial cocktail)

• Extraction procedure:

  • Add 5 ml buffer per gram of tissue

  • Heat samples at 95°C for 5 minutes

  • Centrifuge at 14,000 × g for 10 minutes at 4°C

  • Collect supernatant and determine protein concentration

• SDS-PAGE conditions:

  • Load 10-15 μg total protein per lane

  • Use 12% polyacrylamide gels for optimal resolution

  • Include molecular weight markers

Similar plant antibodies demonstrate optimal results with these methods .

What are the recommended dilution ratios for different applications of At1g31080 antibody?

Based on similar plant antibodies and standard protocols for polyclonal antibodies:

These dilutions should be optimized for each specific experimental setup. For Western blotting, dilute the antibody in TBS-T buffer containing 2-5% non-fat dry milk or BSA. For ELISA, dilute in the appropriate blocking buffer according to your established protocol .

How should I design controls for experiments using At1g31080 antibody?

A methodologically sound experimental design should include these controls:

• Positive control: Include protein extract from wild-type Arabidopsis thaliana known to express At1g31080.

• Negative control: Use one of the following:

  • Protein extract from knockout/knockdown lines of At1g31080 if available

  • Pre-immune serum at the same dilution as the primary antibody

  • Primary antibody pre-absorbed with excess immunizing peptide

• Loading control: Include detection of a constitutively expressed protein such as actin or GAPDH to normalize expression levels.

• Secondary antibody control: Omit primary antibody but include secondary antibody to identify non-specific binding.

These controls help distinguish specific signals from background and validate antibody specificity .

What are the optimal storage conditions for maintaining At1g31080 antibody activity?

For maximum stability and activity retention:

• Long-term storage: Store at -20°C to -80°C in the manufacturer's buffer (typically containing 50% glycerol).

• Working aliquots: Upon receipt, prepare small working aliquots (10-50 μl) to avoid repeated freeze-thaw cycles.

• Thawing procedure: Thaw aliquots on ice, briefly centrifuge before opening tubes to collect all liquid.

• Short-term storage: For ongoing experiments, antibody dilutions can be stored at 4°C for up to one week with the addition of 0.02% sodium azide as preservative.

• Critical precaution: Never store diluted antibody solutions without preservative at 4°C for extended periods as this may lead to microbial contamination and degradation .

How can I troubleshoot high background issues when using At1g31080 antibody?

When encountering high background in Western blots or other applications:

• Optimize antibody dilution: Test a dilution series (e.g., 1:1000, 1:3000, 1:5000, 1:10000) to determine optimal signal-to-noise ratio.

• Improve blocking conditions:

  • Increase blocking time (2-3 hours at room temperature or overnight at 4°C)

  • Test different blocking agents (5% non-fat dry milk, 3-5% BSA, or commercial blocking reagents)

  • Add 0.1-0.3% Tween-20 to blocking and washing buffers

• Optimize washing steps:

  • Increase number of washes (5-6 times for 5-10 minutes each)

  • Use larger volumes of wash buffer

  • Add higher concentrations of Tween-20 (up to 0.1%) or NaCl (up to 500 mM) to washing buffer

• Reduce non-specific binding:

  • Add 1-5% blocking agent to antibody dilution buffer

  • Pre-absorb antibody with plant extract from species that do not express the target

• Reduce detection sensitivity if signal-to-noise ratio remains poor despite optimization .

What is the expected cross-reactivity profile of At1g31080 antibody with other plant species?

The At1g31080 antibody is specifically designed to recognize Arabidopsis thaliana protein. Cross-reactivity with other species depends on sequence conservation of the immunogen region. When working with non-Arabidopsis species:

• Sequence homology analysis: Perform BLAST analysis comparing At1g31080 sequence with the corresponding protein in your species of interest to predict potential cross-reactivity.

• Empirical testing: Always validate cross-reactivity experimentally by:

  • Running Western blots with protein extracts from multiple species

  • Including positive (Arabidopsis) and negative controls

  • Comparing observed band patterns with predicted molecular weights

• Epitope conservation assessment: If the specific epitope sequence is known, compare this region specifically across species for higher predictive value.

Unlike some plant antibodies that show broad cross-reactivity (such as actin antibodies that react with proteins from multiple plant species including Arabidopsis thaliana, Brassica sp., Cucumis sativus, etc.), species-specific antibodies like At1g31080 may have limited cross-reactivity .

How can I use At1g31080 antibody for protein-protein interaction studies?

For investigating protein interactions involving At1g31080:

• Co-immunoprecipitation (Co-IP):

  • Prepare plant extract under native conditions (non-denaturing buffer)

  • Incubate with At1g31080 antibody conjugated to protein A/G beads

  • Isolate protein complexes and analyze interacting partners by mass spectrometry

  • Validate interactions by reverse Co-IP with antibodies against identified partners

• Proximity Ligation Assay (PLA):

  • Fix and permeabilize plant cells/tissues

  • Incubate with At1g31080 antibody and antibody against putative interacting protein

  • Apply species-specific PLA probes and perform rolling circle amplification

  • Analyze interaction signals by fluorescence microscopy

• Bimolecular Fluorescence Complementation (BiFC) validation:

  • Generate fusion constructs of At1g31080 and candidate interactors with split fluorescent protein fragments

  • Express in plant protoplasts or via transient expression

  • Analyze reconstituted fluorescence as indication of protein-protein interaction

These approaches provide complementary evidence for protein interactions in different experimental contexts .

What are the methodological considerations for using At1g31080 antibody in immunolocalization studies?

For successful subcellular localization studies:

• Sample preparation protocol:

  • Fix plant tissue in 4% paraformaldehyde for 45-60 minutes

  • Perform tissue clearing if necessary (e.g., with ClearSee or modified Truant's clearing solution)

  • Permeabilize with 0.1-0.5% Triton X-100

  • Block with 3-5% BSA or normal serum

• Antibody application:

  • Use At1g31080 antibody at 1:100-1:250 dilution

  • Incubate samples overnight at 4°C

  • Apply fluorescently-labeled secondary antibody (1:500 dilution)

  • Include DAPI for nuclear counterstaining

• Imaging optimization:

  • Use confocal microscopy for optimal resolution

  • Collect Z-stacks to capture 3D distribution

  • Include appropriate controls (secondary antibody only, pre-immune serum)

• Super-resolution options:

  • For detailed subcellular localization, consider expansion microscopy (ExM)

  • When using ExM, dilute At1g31080 antibody 1:250

  • Follow standard ExM protocols for sample expansion and imaging

These methods allow precise determination of the subcellular localization of At1g31080 protein .

How can At1g31080 antibody be used to investigate protein modifications and degradation pathways?

To study post-translational modifications and protein degradation:

• Phosphorylation analysis:

  • Treat samples with phosphatase inhibitors during extraction

  • Perform immunoprecipitation with At1g31080 antibody

  • Analyze by Western blot with phospho-specific antibodies

  • Confirm with mass spectrometry phosphopeptide mapping

• Ubiquitination studies:

  • Treat plants with proteasome inhibitors (MG132)

  • Perform immunoprecipitation under denaturing conditions

  • Probe Western blots with both At1g31080 and ubiquitin antibodies

  • Analyze higher molecular weight bands indicating ubiquitinated forms

• Protein stability assays:

  • Treat plants with cycloheximide to inhibit protein synthesis

  • Collect samples at different time points

  • Perform Western blot with At1g31080 antibody

  • Quantify protein levels to determine half-life

• Stress-induced modification profiling:

  • Subject plants to various stresses (drought, salt, heat)

  • Analyze changes in At1g31080 protein abundance, modification state, and localization

  • Compare with transcriptional responses to distinguish regulatory mechanisms

These approaches help elucidate the regulation and turnover of At1g31080 protein under different conditions .

How can I combine At1g31080 antibody detection with transcriptomic analyses?

To correlate protein expression with transcriptional regulation:

• Integrated analysis protocol:

  • Extract RNA and protein from the same tissue samples

  • Perform RT-qPCR or RNA-seq for transcript quantification

  • Use Western blot with At1g31080 antibody for protein detection

  • Normalize protein levels to housekeeping controls (e.g., actin)

  • Compare transcript and protein abundance patterns

• Time-course experimental design:

  • Collect samples at multiple time points after treatment or during development

  • Analyze transcript levels by RT-qPCR

  • Determine protein levels by quantitative Western blotting

  • Calculate correlation coefficients between mRNA and protein levels

  • Identify time lags suggesting post-transcriptional regulation

• Data integration approach:

  • Create scatter plots of transcript vs. protein levels

  • Calculate Pearson or Spearman correlation coefficients

  • Identify conditions with discordant mRNA/protein patterns

  • Investigate potential regulatory mechanisms (e.g., miRNA targeting, protein stability)

This integrated approach reveals post-transcriptional regulation mechanisms affecting At1g31080 expression .

What methodological approaches can be used to validate At1g31080 antibody specificity in research applications?

For rigorous validation of antibody specificity:

• Genetic validation:

  • Test antibody against wild-type and knockout/knockdown lines

  • Verify absence/reduction of signal in mutant lines

  • Complement with overexpression lines to confirm signal increase

• Biochemical validation:

  • Perform peptide competition assay (pre-incubate antibody with immunizing peptide)

  • Compare Western blot patterns with predicted molecular weight

  • Confirm single band at expected size in Arabidopsis samples

• Mass spectrometry validation:

  • Immunoprecipitate using At1g31080 antibody

  • Analyze precipitated proteins by mass spectrometry

  • Confirm presence of At1g31080 peptides

  • Assess presence of potentially cross-reacting proteins

• Recombinant protein control:

  • Express tagged recombinant At1g31080 protein

  • Perform parallel detection with At1g31080 antibody and tag-specific antibody

  • Verify co-localization of signals

How does the methodology for At1g31080 antibody applications compare with other plant protein antibodies?

When comparing methodological approaches:

This comparison helps researchers adapt protocols from well-established antibodies when working with less-characterized ones like At1g31080 .

What are the critical considerations when designing experiments to study protein-protein interactions involving At1g31080?

For robust protein interaction studies:

• Experimental design hierarchy:

  • Begin with in silico prediction of interaction partners

  • Perform co-immunoprecipitation with At1g31080 antibody

  • Validate with orthogonal methods (Y2H, BiFC, FRET)

  • Confirm biological relevance with functional assays

• Buffer optimization critical factors:

  • Salt concentration affects interaction strength (150-300 mM NaCl typical)

  • Detergent type and concentration preserves interactions while solubilizing membranes

  • pH conditions should mimic cellular compartment of interest

  • Divalent cations (Mg²⁺, Ca²⁺) may be required for certain interactions

• Control experimental design:

  • Include non-specific IgG precipitation control

  • Use protein extract from knockout/knockdown lines

  • Perform reverse co-IP with antibodies against putative partners

  • Include RNase/DNase treatment to eliminate nucleic acid-mediated interactions

• Quantitative analysis approach:

  • Use quantitative proteomics with isotope labeling

  • Calculate enrichment ratios relative to control IPs

  • Apply statistical thresholds for significance

  • Validate top candidates individually

These methodological considerations ensure identification of genuine interaction partners while minimizing false positives .

How can emerging antibody technologies enhance research applications for At1g31080 studies?

Advanced methodological approaches include:

• Single-domain antibody development:

  • Generate camelid-derived nanobodies against At1g31080

  • Advantages include smaller size for tissue penetration and epitope access

  • Can be expressed as intrabodies for in vivo studies

  • Methodology involves immunization, phage display, and recombinant expression

• AI-assisted antibody engineering:

  • Apply computational protein design to optimize At1g31080 antibodies

  • Machine learning models like MAGE (Monoclonal Antibody GEnerator) can generate paired heavy-light chain sequences

  • Requires only antigen sequence input

  • Can improve specificity and reduce cross-reactivity

• Proximity-dependent labeling applications:

  • Fusion of TurboID or APEX2 to At1g31080

  • Enables identification of proximal proteins in native context

  • Complements traditional antibody-based interaction studies

  • Provides spatial and temporal resolution of protein neighborhoods

These emerging technologies expand the methodological toolkit for studying At1g31080 protein function and interactions in plant systems .

What methodological considerations should be addressed when designing experiments to study At1g31080 function under stress conditions?

For robust stress response studies:

• Experimental design framework:

  • Include appropriate time course (immediate, short-term, long-term responses)

  • Apply graduated stress intensities to identify thresholds

  • Compare multiple stress types (drought, salt, heat, pathogen)

  • Include recovery phase to assess reversibility

• Sample preparation optimization:

  • Harvest tissues at consistent time of day (control for circadian effects)

  • Flash-freeze samples immediately to preserve modification state

  • Extract in phosphatase/protease inhibitor-containing buffers

  • Process all samples simultaneously to minimize batch effects

• Analytical approach:

  • Quantify both total protein (standard Western blot) and modified forms

  • Perform subcellular fractionation to detect translocation events

  • Compare transcript and protein levels to identify post-transcriptional regulation

  • Use phospho-specific antibodies if phosphorylation sites are known

• Genetic approach integration:

  • Compare wild-type, knockout, and overexpression lines

  • Assess phenotypic differences under stress conditions

  • Perform transcriptome analysis to identify affected pathways

  • Use complementation studies to confirm functional relationships

This comprehensive approach allows researchers to determine the role of At1g31080 in stress responses with methodological rigor .

What are the best practices for reporting At1g31080 antibody usage in scientific publications?

For reproducible research reporting:

• Essential antibody information to include:

  • Complete antibody identifier (catalog number, clone, lot)

  • Host species and clonality

  • Immunogen details (full sequence if available)

  • Supplier and validation performed

  • RRID (Research Resource Identifier) if available

• Methodological details to report:

  • Exact dilutions used for each application

  • Incubation conditions (time, temperature, buffer composition)

  • Detection method specifications (secondary antibody, visualization system)

  • Image acquisition parameters

  • Quantification methods with statistical analysis

• Validation data to include:

  • Positive and negative control results

  • Full blot images with molecular weight markers

  • Specificity controls (peptide competition, genetic validation)

  • Representative images showing subcellular localization

These reporting standards ensure experimental reproducibility and proper evaluation of research findings involving At1g31080 antibody .

What are the methodological priorities for advancing At1g31080 research in plant biology?

Key methodological priorities include:

• Development of comprehensive validation resources:

  • Generation and characterization of knockout/knockdown lines

  • Production of epitope-tagged transgenic lines

  • Creation of phospho-specific antibodies if phosphorylation sites are identified

  • Establishment of standardized protocols specific to At1g31080

• Integration with emerging technologies:

  • Application of proximity labeling approaches (BioID, APEX)

  • Implementation of super-resolution microscopy techniques

  • Development of CRISPR-based tools for endogenous tagging

  • Application of quantitative proteomics for interaction studies

• Functional characterization priorities:

  • Systematic phenotypic analysis under various conditions

  • Determination of subcellular localization and dynamics

  • Identification of post-translational modifications

  • Elucidation of protein-protein interaction networks