At3g23880 Antibody

What is At3g23880 and what role does it play in Arabidopsis thaliana?

At3g23880 is a gene locus on chromosome 3 of Arabidopsis thaliana, which encodes a protein involved in plant stress responses. Based on sequence homology analysis, it belongs to a family of proteins that participate in cellular signaling pathways. Gene expression studies have shown that At3g23880 expression changes in response to various environmental stressors, similar to other stress-responsive genes such as WRKY6, which is involved in arsenate response pathways . Understanding the function of this protein requires specific antibodies for detection and characterization in experimental systems.

How are antibodies against plant proteins like At3g23880 typically generated?

Antibodies against plant proteins like At3g23880 are typically generated through the following methodological approaches:

• Recombinant protein expression: The coding sequence of At3g23880 is cloned into an expression vector, expressed in E. coli or other host systems, and purified for immunization.

• Synthetic peptide design: Specific immunogenic peptide sequences (15-20 amino acids) unique to At3g23880 are identified through bioinformatic analysis, synthesized, and conjugated to carrier proteins like KLH (keyhole limpet hemocyanin).

• Immunization protocols: Rabbits or mice are typically immunized with the antigen following a prime-boost schedule over 2-3 months. For monoclonal antibodies, B cells from immunized mice are isolated and fused with myeloma cells to produce hybridomas.

• Antibody screening and purification: Antibodies are screened for specificity and sensitivity using ELISA, Western blotting, or other immunoassays against the target protein and purified using affinity chromatography.

The selection of target epitopes is critical for antibody specificity, as demonstrated in various immunological studies of plant proteins .

What are the recommended sample preparation methods for At3g23880 antibody applications?

Sample preparation methods should be optimized based on the specific application and plant tissue type:

For At3g23880 in particular, protein extraction from Arabidopsis tissues should include steps to remove phenolic compounds and other plant-specific interfering substances, which can significantly improve antibody binding specificity.

How can At3g23880 antibody be used to study protein localization during stress responses?

At3g23880 antibody can be used for subcellular localization studies to track protein redistribution during stress responses through these methodological approaches:

• Immunofluorescence microscopy: Fix plant tissues or protoplasts with paraformaldehyde, permeabilize with Triton X-100, and incubate with At3g23880 primary antibody followed by fluorescently-labeled secondary antibody. Counter-stain with organelle markers to determine precise localization.

• Subcellular fractionation and Western blotting: Fractionate plant cells into cytoplasmic, nuclear, membrane, and organelle fractions, then perform Western blotting with At3g23880 antibody to track protein redistribution under different stress conditions.

• Live cell imaging: Generate GFP-tagged At3g23880 and validate localization patterns using the antibody in fixed cells to confirm that the tagged protein behaves like the endogenous protein.

This approach has been successfully used to track proteins like PHT1;1 in Arabidopsis, which showed dynamic changes in localization in response to arsenite treatment, moving from the plasma membrane to internal vesicles within 6 hours of treatment .

What controls are essential when using At3g23880 antibody for experimental validation?

Researchers must implement the following controls to ensure experimental validity:

As demonstrated in studies of other Arabidopsis proteins, the use of actin as a loading control provides reliable normalization for quantitative analysis, as seen in experiments tracking PHT1;1 protein levels where actin hybridization was used as a loading control .

How do you analyze potential cross-reactivity of At3g23880 antibody with related proteins?

Cross-reactivity analysis requires a systematic approach:

• Bioinformatic prediction: Use BLAST analysis to identify proteins with similar epitope sequences in Arabidopsis.

• Recombinant protein testing: Express and purify proteins with similar sequences and test antibody binding by Western blot.

• Knockout validation: Test antibody specificity in At3g23880 knockout or knockdown lines, which should show reduced or no signal.

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to identify all proteins captured by the antibody.

• Epitope mapping: Determine the exact binding site of the antibody using peptide arrays or truncated protein variants.

What are the optimal Western blotting conditions for At3g23880 antibody?

Optimized Western blotting conditions for At3g23880 antibody typically include:

• Sample preparation: Extract total protein using buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail.

• Gel electrophoresis: Separate 20-50 μg of protein on 10-12% SDS-PAGE gels at 100V until the dye front reaches the bottom.

• Transfer conditions: Transfer to PVDF membrane at 100V for 1 hour in cold transfer buffer or 30V overnight at 4°C.

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

• Antibody incubation:

  • Primary antibody: Dilute At3g23880 antibody 1:1000 in 5% BSA in TBST, incubate overnight at 4°C

  • Secondary antibody: Anti-rabbit HRP at 1:5000 in 5% milk in TBST for 1 hour at room temperature

• Detection: Use ECL substrate and expose to X-ray film or digital imager.

These conditions should be optimized for each new antibody batch, as sensitivity can vary. Similar proteins like PHT1;1 have been successfully detected using immunoblot analyses with specific optimization of transfer and blocking conditions .

How can At3g23880 antibody be used for co-immunoprecipitation to identify protein interaction partners?

Co-immunoprecipitation (Co-IP) with At3g23880 antibody can be performed using this methodological approach:

• Sample preparation:

  • Harvest 5-10 g of Arabidopsis tissue and grind in liquid nitrogen

  • Extract proteins in IP buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 3 mM DTT, protease inhibitors)

  • Clarify by centrifugation at 14,000 × g for 15 minutes at 4°C

• Pre-clearing:

  • Incubate lysate with Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Add At3g23880 antibody (5-10 μg) to pre-cleared lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add 50 μl Protein A/G beads and incubate for 3 hours at 4°C

  • Wash beads 5 times with IP buffer

• Elution and analysis:

  • Elute proteins with SDS sample buffer by heating at 95°C for 5 minutes

  • Analyze by SDS-PAGE followed by Western blotting or mass spectrometry

This approach can identify protein complexes involving At3g23880, similar to how protein interactions have been studied in other plant systems to understand signaling pathways and protein function networks.

What quantitative methodologies can be used with At3g23880 antibody for protein expression analysis?

Several quantitative methods can be employed with At3g23880 antibody:

The choice of method depends on research objectives, sample availability, and required sensitivity. For example, ELISA would be preferred for precise quantification across many samples, while immunohistochemistry provides spatial information at the expense of precise quantification.

How do you reconcile discrepancies between At3g23880 transcript levels and protein abundance?

Discrepancies between transcript and protein levels for At3g23880 should be analyzed through:

• Post-transcriptional regulation assessment: Investigate microRNA targeting, RNA stability, and alternative splicing that might affect mRNA translation efficiency.

• Protein stability analysis: Measure protein half-life using cycloheximide chase assays to determine if differences stem from protein degradation rates rather than synthesis.

• Temporal resolution studies: Perform time-course experiments to detect potential time lags between transcription and translation, as observed in stress response systems.

• Translational efficiency evaluation: Use polysome profiling to assess whether At3g23880 mRNA is efficiently loaded onto ribosomes for translation.

• Protein modification tracking: Investigate post-translational modifications that might affect antibody recognition or protein stability.

Research on other Arabidopsis genes has shown that arsenic stress can cause rapid changes in protein localization without immediate degradation, as observed with PHT1;1, which showed membrane internalization within 3-6 hours of arsenite treatment while protein levels remained relatively stable .

What experimental designs can elucidate the functional role of At3g23880 in stress response pathways?

Comprehensive experimental designs to investigate At3g23880 function include:

• Expression profiling under various stresses:

  • Treat Arabidopsis plants with different stressors (drought, salt, heat, cold, pathogens)

  • Monitor At3g23880 protein levels using quantitative Western blotting

  • Compare with transcript levels via qRT-PCR

  • Similar studies with arsenate stress showed upregulation of genes like WRKY6 within 3 hours of treatment

• Genetic manipulation studies:

  • Generate knockout/knockdown lines of At3g23880 using T-DNA insertion or CRISPR-Cas9

  • Create overexpression lines with constitutive promoters

  • Assess phenotypic changes under normal and stress conditions

  • Measure downstream signaling component alterations

• Protein localization dynamics:

  • Track protein redistribution using immunofluorescence microscopy

  • Correlate relocalization with stress response timing

  • Similar to studies showing PHT1;1-GFP internalization in vesicles after arsenite treatment

• Interactome analysis:

  • Perform co-immunoprecipitation followed by mass spectrometry

  • Validate key interactions with yeast two-hybrid or bimolecular fluorescence complementation

  • Map interaction networks under different stress conditions

These multi-faceted approaches can reveal both direct mechanistic roles and broader pathway involvements of At3g23880 in plant stress responses.

How can phosphorylation status of At3g23880 be determined using phospho-specific antibodies?

Phosphorylation status determination requires specific methodological approaches:

• Phosphorylation site prediction:

  • Use bioinformatic tools (NetPhos, PhosphoSite, etc.) to predict potential phosphorylation sites

  • Focus on serine, threonine, and tyrosine residues in conserved motifs

  • Design phospho-specific antibodies against these sites

• Phospho-specific antibody generation:

  • Synthesize phosphopeptides corresponding to predicted phosphorylation sites

  • Generate antibodies that specifically recognize phosphorylated epitopes

  • Purify using affinity chromatography against phospho and non-phospho peptides

• Validation of phospho-specific antibodies:

  • Test specificity using in vitro phosphorylated recombinant protein

  • Confirm with lambda phosphatase treatment to remove phosphorylation

  • Include phosphorylation-site mutants (S/T/Y to A) as negative controls

• Application to biological samples:

  • Extract proteins under phosphatase-inhibiting conditions

  • Perform Western blotting with both phospho-specific and total protein antibodies

  • Calculate phosphorylation ratio (phospho-specific signal/total protein signal)

This approach has been used successfully to study phosphorylation-dependent regulation of plant proteins involved in stress signaling pathways, including those regulated by arsenite, which has been shown to function as a signaling molecule affecting protein stability and localization .

What are common challenges when using plant protein antibodies and how can they be addressed?

Common challenges with plant protein antibodies can be addressed through specific strategies:

Plant tissues present unique challenges due to high levels of phenolics, polysaccharides, and proteases. Adding PVPP (polyvinylpolypyrrolidone) to extraction buffers can help remove phenolic compounds that interfere with antibody binding .

How do you optimize immunohistochemistry protocols for At3g23880 localization in plant tissues?

Optimization of immunohistochemistry for At3g23880 localization requires:

• Fixation optimization:

  • Test different fixatives: 4% paraformaldehyde, Carnoy's solution, or glutaraldehyde

  • Optimize fixation time (2-24 hours) and temperature

  • Include vacuum infiltration steps for better penetration in plant tissues

• Antigen retrieval methods:

  • Heat-induced epitope retrieval: citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0)

  • Enzymatic retrieval: proteinase K, trypsin, or pepsin treatment

  • Determine optimal treatment duration for your specific tissue

• Permeabilization and blocking optimization:

  • Test detergent concentrations (0.1-1% Triton X-100)

  • Compare blocking agents: BSA, normal serum, or commercial blockers

  • Evaluate blocking times (1-24 hours)

• Antibody incubation parameters:

  • Titrate antibody dilutions (1:100 to 1:2000)

  • Test incubation times (overnight to 48 hours) and temperatures (4°C, RT)

  • Consider using amplification systems for low-abundance proteins

• Signal development optimization:

  • Compare DAB, fluorescent secondary antibodies, or tyramide signal amplification

  • Determine optimal development time for each system

  • Include appropriate controls for autofluorescence (common in plant tissues)

Similar approaches have been applied to localization studies of membrane transporters like PHT1;1, where specific conditions were required to visualize membrane-associated versus internalized protein pools .