At1g14315 Antibody

What is the At1g14315 gene and its corresponding protein in Arabidopsis thaliana?

At1g14315 is a gene locus in Arabidopsis thaliana (Mouse-ear cress) that encodes protein Q9M9T0, which appears to be functionally related to S-locus F-box proteins . These proteins are involved in self-incompatibility mechanisms in plants, regulating pollen recognition and fertilization processes. The gene is part of a larger network of proteins that maintain reproductive diversity in plant populations. While its complete characterization is still evolving, current research indicates its importance in plant reproductive biology and potentially in stress response pathways.

What are the primary research applications of the At1g14315 Antibody?

The At1g14315 Antibody (commercially available as CSB-PA881938XA01DOA) serves multiple research purposes in plant molecular biology :

These applications enable researchers to investigate the functional role of the At1g14315 protein in development, stress responses, and reproductive pathways.

How does the specificity of At1g14315 Antibody compare to antibodies for other Arabidopsis proteins?

The specificity of At1g14315 Antibody reflects the highly selective nature of modern plant antibody production. Similar to antibodies developed against other Arabidopsis proteins (like At1g71320, At1g70390, and others in the same catalog), this antibody undergoes rigorous validation to ensure minimal cross-reactivity . The antibody binds specifically to the target epitope region of the At1g14315-encoded protein without significant binding to homologous proteins. This specificity results from careful antigen design, typically targeting unique regions of the protein that have low sequence homology with other plant proteins.

What are the recommended protocols for validating At1g14315 Antibody specificity before experimental use?

Comprehensive validation of At1g14315 Antibody specificity should include multiple complementary approaches:

• Western Blot with Positive and Negative Controls:

  • Use wild-type Arabidopsis tissue as positive control

  • Use At1g14315 knockout/knockdown lines as negative control

  • Verify single band at expected molecular weight (~predicted kDa for Q9M9T0)

• Peptide Competition Assay:

  • Pre-incubate antibody with 5-10× excess of immunizing peptide

  • Perform parallel Western blots with blocked and unblocked antibody

  • Expect signal elimination in the blocked condition

• Immunoprecipitation Followed by Mass Spectrometry:

  • Verify pulled-down protein identity via MS/MS analysis

  • Compare detected peptides against Arabidopsis protein database

This validation approach mirrors established protocols for other research antibodies where specificity is paramount for accurate data interpretation .

What cross-reactivity concerns should researchers address when using At1g14315 Antibody?

When working with plant antibodies like At1g14315 Antibody, researchers should systematically address potential cross-reactivity issues:

• Homologous Protein Cross-Reactivity: Test against closely related F-box family proteins, particularly those with similar epitope regions.

• Species Cross-Reactivity: While designed for Arabidopsis thaliana, determine if the antibody recognizes homologous proteins in related plant species when performing comparative studies .

• Non-specific Binding Assessment: Perform the following controls:

  • Pre-immune serum control

  • Secondary antibody-only control

  • Testing in tissues known to lack the target protein

Similar to validation approaches used for other research antibodies, including those against receptor proteins, ensuring minimal cross-reactivity is essential for confident interpretation of experimental results .

What are the optimal sample preparation methods for Western blotting with At1g14315 Antibody?

For optimal Western blot detection of the At1g14315 protein, the following sample preparation protocol is recommended:

• Tissue Extraction Buffer:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 0.5% Sodium deoxycholate

  • Plant protease inhibitor cocktail

  • 1 mM PMSF

  • 5 mM DTT

• Critical Sample Handling Steps:

  • Harvest tissue quickly and flash-freeze in liquid nitrogen

  • Grind tissue to fine powder while maintaining frozen state

  • Use 4:1 buffer-to-tissue ratio (v/w)

  • Centrifuge at 14,000×g for 15 minutes at 4°C

  • Collect supernatant and quantify protein concentration

• Protein Denaturation:

  • Mix samples with 4× Laemmli buffer

  • Heat at 95°C for 5 minutes

  • Load 20-40 μg total protein per lane

This methodology ensures preservation of protein integrity and epitope accessibility, similar to approaches used for other plant proteins where maintaining native conformation is important prior to denaturation .

How can At1g14315 Antibody be effectively employed in chromatin immunoprecipitation (ChIP) studies?

For chromatin immunoprecipitation studies investigating potential DNA-protein interactions of At1g14315, researchers should implement this optimized protocol:

• Crosslinking and Chromatin Preparation:

  • Crosslink fresh plant tissue with 1% formaldehyde for 10 minutes under vacuum

  • Quench with 0.125 M glycine for 5 minutes

  • Extract nuclei using plant nuclei isolation buffer (0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl₂, 1% Triton X-100, 5 mM β-mercaptoethanol, protease inhibitors)

  • Sonicate chromatin to generate 200-500 bp fragments

• Immunoprecipitation Parameters:

  • Use 5-10 μg of At1g14315 Antibody per reaction

  • Include IgG control antibody at equivalent concentration

  • Pre-clear chromatin with protein A/G beads

  • Incubate antibody-chromatin mixture overnight at 4°C

  • Wash stringently with increasingly stringent buffers

• DNA Recovery and Analysis:

  • Reverse crosslinks at 65°C for 6 hours

  • Treat with proteinase K and RNase A

  • Purify DNA using silica column-based methods

  • Perform qPCR or sequencing to identify binding regions

This approach parallels methodologies established for other DNA-binding proteins, enabling investigation of potential chromatin-associated functions of At1g14315 .

What approaches should be used to resolve contradictory results with At1g14315 Antibody across different experimental conditions?

When confronted with contradictory results using At1g14315 Antibody, implement this systematic troubleshooting framework:

• Antibody Validation Reassessment:

  • Repeat specificity testing with fresh antibody aliquots

  • Test different antibody lots if available

  • Consider alternative antibodies targeting different epitopes of the same protein

• Sample-Specific Variables Analysis:

  • Systematically document growth conditions (light cycle, temperature, medium)

  • Record plant developmental stages with precision

  • Control for stress exposure prior to sampling

• Technical Parameter Optimization Matrix:

  • Test a range of antibody concentrations (0.1-10 μg/mL)

  • Vary blocking conditions (5% milk vs. 3% BSA)

  • Adjust incubation times and temperatures

• Comparative Analysis Approach:

  • Implement parallel detection with orthogonal methods (e.g., fluorescent protein tagging)

  • Correlate protein detection with transcript levels (RT-qPCR)

  • Validate findings in multiple biological replicates across seasonal variations

This structured approach to resolving contradictory results follows established practices in immunological research where variability can emerge from multiple sources .

How can researchers effectively combine At1g14315 Antibody with mass spectrometry for protein interaction studies?

For comprehensive protein interaction studies combining immunoprecipitation with mass spectrometry:

• Optimized Immunoprecipitation Protocol:

  • Extract proteins under native conditions (avoid harsh detergents)

  • Use 5 μg At1g14315 Antibody conjugated to magnetic protein A/G beads

  • Include appropriate negative controls (IgG, knockout lines)

  • Wash with buffers of decreasing stringency to preserve interactions

  • Elute protein complexes with gentle, MS-compatible methods

• Sample Preparation for MS Analysis:

  • Perform on-bead or in-gel tryptic digestion

  • Implement a filter-aided sample preparation (FASP) protocol

  • Fractionate peptides using basic reversed-phase chromatography

  • Label samples with TMT or iTRAQ for quantitative comparison

• Data Analysis Pipeline:

  • Search against Arabidopsis thaliana protein database

  • Filter protein identifications (1% FDR threshold)

  • Implement SAINT or CRAPome algorithms to distinguish true interactors from background

  • Validate key interactions via reciprocal pulldowns

This methodology enables identification of protein interaction networks, similar to approaches used in other complex biological systems where specific antibodies facilitate isolation of protein complexes .

How can At1g14315 Antibody be integrated with super-resolution microscopy for advanced localization studies?

For high-resolution subcellular localization of At1g14315 protein:

• Sample Preparation for Super-Resolution Microscopy:

  • Fix Arabidopsis seedlings or leaves with 4% paraformaldehyde

  • Permeabilize cell walls with 0.1% Driselase followed by 0.5% Triton X-100

  • Block with 3% BSA in PBS for 1 hour

  • Incubate with At1g14315 Antibody (1:100) overnight at 4°C

  • Use fluorophore-conjugated secondary antibodies optimized for super-resolution

• Imaging Parameters for Different Super-Resolution Techniques:

• Data Acquisition and Analysis:

  • Collect Z-stacks to capture 3D distribution

  • Implement drift correction using fiducial markers

  • Process images with technique-specific reconstruction algorithms

  • Perform colocalization analysis with organelle markers

This approach enables nanoscale visualization of protein distribution patterns that conventional microscopy cannot resolve, similar to advanced imaging techniques applied to other complex biological systems .

What are the considerations for generating custom phospho-specific antibodies against At1g14315 protein?

For researchers interested in developing phospho-specific antibodies to study At1g14315 regulation:

• Phosphorylation Site Prediction and Selection:

  • Analyze At1g14315 sequence using PhosphoSitePlus and NetPhos

  • Prioritize evolutionarily conserved sites

  • Consider sites in functional domains or near protein interaction motifs

  • Validate predicted sites using phosphoproteomics data if available

• Peptide Design Strategy:

  • Design 10-15 amino acid peptides centered on phosphorylation site

  • Include phosphorylated residue (pSer, pThr, or pTyr)

  • Add C-terminal cysteine for carrier protein conjugation

  • Consider multiple peptides per phosphorylation site

• Production and Validation Protocol:

  • Immunize rabbits with phosphopeptide conjugated to KLH

  • Collect serum and purify antibodies using dual affinity approach:

    • Positive selection with phosphopeptide column

    • Negative selection with non-phosphopeptide column

  • Validate specificity using:

    • Peptide arrays with phospho and non-phospho peptides

    • Western blots comparing phosphatase-treated vs. untreated samples

    • Knockout/knockdown line controls

This methodological approach parallels established strategies for developing other post-translational modification-specific antibodies in research settings .

How can CRISPR/Cas9 gene editing be used to validate At1g14315 Antibody specificity in Arabidopsis?

For definitive validation of At1g14315 Antibody specificity using gene editing:

• CRISPR/Cas9 Knockout Strategy:

  • Design sgRNAs targeting early exons of At1g14315

  • Introduce frameshift mutations to ensure complete protein loss

  • Generate homozygous knockout lines through segregation

  • Confirm editing by sequencing and transcript analysis

• Epitope Modification Approach:

  • Design sgRNAs targeting the region encoding the antibody epitope

  • Implement HDR to introduce specific amino acid changes within the epitope

  • Generate lines with modified but functional At1g14315 protein

• Comprehensive Validation Protocol:

  • Perform side-by-side Western blot analysis:

    • Wild-type plants (positive control)

    • Complete knockout plants (negative control)

    • Epitope-modified plants (specificity control)

  • Implement immunohistochemistry and immunofluorescence comparisons

  • Quantify signal-to-noise ratios across all genotypes

This gene editing validation approach provides the most definitive assessment of antibody specificity, creating biological controls that are impossible to generate through other means .

What strategies can address weak or inconsistent signals when using At1g14315 Antibody?

When encountering weak or inconsistent signals, implement this hierarchical troubleshooting approach:

• Sample Preparation Optimization:

  • Increase protein extraction efficiency with modified buffers:

    • Add 0.1% SDS to standard extraction buffer

    • Include 6M urea for difficult-to-extract proteins

    • Test sonication vs. mechanical disruption methods

  • Implement protease inhibitor cocktails optimized for plant tissues

  • Avoid freeze-thaw cycles of protein samples

• Antibody Handling and Protocol Adjustments:

  • Test various antibody concentrations (0.5-5 μg/mL)

  • Extend primary antibody incubation (overnight at 4°C)

  • Implement signal enhancement systems:

    • Biotin-streptavidin amplification

    • Tyramide signal amplification (TSA)

    • Enhanced chemiluminescent substrates

• Protein Expression Modulation:

  • Apply treatments known to upregulate At1g14315 expression

  • Target tissues/developmental stages with highest expression

  • Consider concentrating proteins via immunoprecipitation prior to detection

This systematic approach addresses the multiple variables that can impact antibody performance in plant research applications .

How should researchers evaluate batch-to-batch variation in At1g14315 Antibody performance?

To systematically assess and mitigate batch-to-batch variation:

• Standardized Quality Control Protocol:

  • Perform side-by-side Western blot comparison with:

    • Previous antibody batch at equivalent concentration

    • Standardized positive control lysate (aliquoted and stored at -80°C)

  • Quantify key performance metrics:

    • Signal-to-noise ratio

    • EC₅₀ value in dilution series

    • Band intensity at standardized exposure

• Performance Documentation System:

  • Maintain detailed records including:

    • Lot number and production date

    • Dilution factor and incubation conditions

    • Detection method and exposure times

    • Raw image files with standardized processing

• Reference Standard Implementation:

  • Create stable reference samples:

    • Lyophilized plant extracts with known At1g14315 expression

    • Recombinant protein standards at defined concentrations

  • Use reference standards with each new experiment

This structured evaluation approach enables reliable comparison across experiments and antibody batches, similar to quality control practices in other areas of biological research .

How might monoclonal antibody development technologies improve At1g14315 research?

The development of monoclonal antibodies for At1g14315 represents a significant advancement opportunity:

• Benefits of Transitioning to Monoclonal Antibodies:

  • Enhanced reproducibility through defined epitope targeting

  • Elimination of polyclonal batch-to-batch variation

  • Improved specificity with single epitope recognition

  • Potential for renewable antibody source

• Modern Production Technologies:

  • Phage display selection from synthetic libraries

  • Single B-cell isolation and antibody cloning

  • Recombinant antibody expression in plant systems

  • AI-guided epitope selection for optimal specificity

• Implementation Strategy for Plant Research:

  • Target conserved epitopes to enable cross-species application

  • Develop paired antibodies recognizing distinct epitopes

  • Create tagged recombinant versions for specialized applications

  • Validate in multiple Arabidopsis ecotypes

The transition to monoclonal antibodies would parallel developments in other research fields where precise epitope targeting enhances experimental reproducibility and data reliability .

What are the considerations for using At1g14315 Antibody in plant species beyond Arabidopsis thaliana?

For cross-species applications of At1g14315 Antibody:

• Sequence Homology Assessment:

  • Perform bioinformatic analysis of epitope conservation across:

    • Close relatives (Brassicaceae family)

    • Other model plants (rice, tomato, maize)

    • Evolutionary distant species if relevant

  • Calculate percent identity and similarity at epitope region

• Validation Protocol for New Species:

  • Implement Western blot with predicted molecular weight adjustments

  • Include positive control (Arabidopsis extract)

  • Perform peptide competition assays

  • Consider immunoprecipitation followed by mass spectrometry

• Protocol Optimization Guidelines:

This cross-species approach enables comparative studies while acknowledging the limitations and required validations when working beyond the original target species .

How can computational approaches improve epitope prediction and antibody design for At1g14315?

Advanced computational tools offer opportunities to enhance At1g14315 Antibody design:

• AI-Driven Epitope Prediction:

  • Implement machine learning algorithms trained on validated plant antibody epitopes

  • Analyze At1g14315 protein structure (predicted or experimental)

  • Score potential epitopes based on:

    • Surface accessibility

    • Secondary structure stability

    • Evolutionary conservation

    • Hydrophilicity profiles

• In silico Antibody Design and Optimization:

  • Apply protein-protein docking simulations

  • Predict binding affinity and specificity

  • Optimize complementarity-determining regions (CDRs)

  • Model potential cross-reactivity with homologous proteins

• Implementation in Research Pipeline:

  • Generate multiple candidate antibodies in silico

  • Produce small-scale test batches of top candidates

  • Implement high-throughput screening against plant proteome arrays

  • Select lead candidates for scaled production

These computational approaches represent the frontier of antibody development, potentially enabling more precise and effective research tools for plant molecular biology .