At1g66250 Antibody

What is AT1G66250 and why is it significant in plant research?

AT1G66250 is a gene encoding Glucan endo-1,3-beta-glucosidase 2, an enzyme that belongs to the beta-glucosidase (BG) family in plants. This protein is particularly significant because it has been confirmed to be located in plasmodesmata (PD), the intercellular channels that allow communication between plant cells . Unlike some other beta-glucosidases that show elevated expression in megaspore mother cells (MMC), AT1G66250 demonstrates specific expression in the nucellus and other somatic tissues surrounding reproductive structures . This expression pattern makes it an important marker for understanding cell-specific functions and symplastic connectivity in plant reproduction and development.

What species reactivity can be expected with the AT1G66250 antibody?

The AT1G66250 antibody has confirmed reactivity with several plant species, primarily Arabidopsis thaliana (Mouse-ear cress). Additional cross-reactivity has been documented with Brassica napus, Brassica rapa, Populus trichocarpa, Cucumis sativus, Spinacia oleracea, and Solanum tuberosum . This broad cross-reactivity among diverse plant species makes the antibody valuable for comparative studies across different plant families, particularly for researchers investigating conserved functions of beta-glucosidases in various plant developmental contexts.

What are the recommended storage conditions for AT1G66250 antibody?

For optimal performance and longevity, the AT1G66250 antibody should be stored at -20°C or -80°C immediately upon receipt . The antibody is typically supplied in a storage buffer containing 50% glycerol, 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative . It's important to avoid repeated freeze-thaw cycles as this can degrade antibody quality and affect binding efficiency . For ongoing experiments, small aliquots can be prepared to minimize freeze-thaw cycles. When using the lyophilized form, reconstitute according to manufacturer instructions and store working solutions at 4°C for short-term use (up to one week).

What basic applications have been validated for the AT1G66250 antibody?

The AT1G66250 antibody has been validated for several standard immunological techniques, with ELISA (Enzyme-Linked Immunosorbent Assay) and Western blot (WB) being the primary confirmed applications . These techniques allow researchers to detect and quantify the presence of AT1G66250 protein in plant tissue extracts. The antibody is supplied as a liquid in non-conjugated form, making it versatile for various detection methods when paired with appropriate secondary antibodies or detection systems. For optimal results in Western blotting, researchers should consider sample preparation methods that effectively solubilize membrane-associated proteins, as AT1G66250 is associated with plasmodesmata membranes.

How can AT1G66250 antibody be used to study plasmodesmata function in reproductive development?

Recent research has revealed that AT1G66250 is specifically expressed in nucellus cells and is confirmed to be located in plasmodesmata (PD) . This localization makes the antibody a valuable tool for studying plasmodesmatal regulation during reproductive development. To investigate PD function using this antibody, researchers can:

• Perform co-immunolocalization studies with other PD markers to examine the spatial organization of different PD components

• Combine with callose staining (using aniline blue) to correlate beta-glucosidase presence with callose deposits

• Use in comparative studies between wild-type and reproductive mutants to assess changes in PD composition

• Implement in developmental time-course experiments to track changes in PD composition during megasporogenesis

These approaches can help elucidate how symplastic connectivity is regulated during female germline specification, which recent studies indicate involves beta-1,3-glucan deposits .

What is the relationship between AT1G66250 and other beta-glucosidase/callose synthase genes?

Transcriptomic analysis of Arabidopsis ovule cells has revealed interesting expression patterns of beta-glucosidases (BGs) and glucan synthase-like (GSL) genes. While six out of nine expressed GSLs (including GSL1, GSL5, GSL8, and GSL10) showed abundant expression in megaspore mother cells (MMCs), AT1G66250 and most other detected BGs showed specific expression in nucellus and surrounding somatic tissues rather than in MMCs . This differential expression suggests a tissue-specific division of labor, where different cell types utilize specific BGs and GSLs to regulate callose deposition and degradation.

The following table summarizes the contrasting expression patterns observed in reproductive tissues:

This expression pattern suggests that AT1G66250 may play specialized roles in maintaining the integrity of plasmodesmata in somatic tissues surrounding reproductive cells, potentially regulating symplastic isolation during germline development .

How does AT1G66250 contribute to symplastic connectivity regulation?

AT1G66250 is confirmed to be located in plasmodesmata and is specifically expressed in nucellus cells surrounding the megaspore mother cell (MMC) . As a beta-1,3-glucosidase, it likely functions in the degradation of callose (beta-1,3-glucan), which is known to regulate the permeability of plasmodesmata. The targeted localization of AT1G66250 suggests a role in fine-tuning symplastic connectivity between somatic cells, while potentially maintaining symplastic isolation of the germline.

Recent research indicates that proper regulation of symplastic connectivity is essential for female reproductive development, with beta-1,3-glucan deposits playing a critical role . The specific expression of AT1G66250 in nucellus cells, rather than in the MMC itself, suggests it may be involved in:

• Maintaining appropriate callose levels at PD in somatic tissues

• Creating symplastic domains during ovule development

• Facilitating selective molecular trafficking between nucellus cells

• Potentially preventing unregulated movement of molecules into the developing germline

These functions make AT1G66250 antibody a valuable tool for studying the molecular mechanisms of cell-to-cell communication during plant reproduction.

What are the optimal sample preparation methods for detecting AT1G66250 in plant tissues?

When designing experiments to detect AT1G66250 in plant tissues, researchers should consider the following protocol recommendations:

• Tissue fixation: Use 4% paraformaldehyde for immunolocalization studies to preserve protein structure while maintaining cellular architecture.

• Protein extraction for Western blotting:

  • Grind tissue in liquid nitrogen

  • Extract with buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitors

  • Include membrane solubilization steps (as AT1G66250 is associated with plasmodesmata)

  • Centrifuge at 12,000 × g for 15 minutes at 4°C

  • Collect supernatant and quantify protein concentration

• Dilution ratios: For Western blotting, start with 1:500 to 1:1000 dilution of the antibody. For ELISA applications, initial testing at 1:1000 to 1:2000 is recommended .

• Controls: Always include appropriate controls:

  • Positive control: Arabidopsis thaliana wild-type tissues with known expression

  • Negative control: Either knockout/knockdown lines for AT1G66250 or tissues where the protein is not expressed

  • Secondary antibody-only control to assess non-specific binding

• Detection systems: For Western blotting, HRP-conjugated secondary antibodies with enhanced chemiluminescence (ECL) provide sensitive detection. For immunofluorescence microscopy, fluorophore-conjugated secondary antibodies compatible with existing filter sets are recommended.

How can AT1G66250 antibody be incorporated into cell-specific transcriptomic studies?

Recent advances in cell-specific transcriptomics have enabled detailed analysis of gene expression in distinct cell types within the Arabidopsis ovule . To incorporate AT1G66250 antibody into such studies, researchers can:

• Use for validation of transcriptomic findings: When RNA-seq data indicates differential expression of AT1G66250, the antibody can confirm protein-level changes through immunolocalization or Western blotting.

• Combine with fluorescence-activated cell sorting (FACS):

  • Fix and permeabilize protoplasts from target tissues

  • Immunostain with AT1G66250 antibody and fluorophore-conjugated secondary antibody

  • Sort cells based on antibody fluorescence

  • Extract RNA from sorted populations for transcriptomic analysis

• Implement in spatial transcriptomics workflows:

  • Perform immunolocalization with AT1G66250 antibody on tissue sections

  • Capture images to document protein localization

  • Process adjacent sections for spatial transcriptomics

  • Correlate protein localization with spatial gene expression patterns

This approach has proven valuable in distinguishing cell-type specific expression profiles in Arabidopsis ovules, where clear separation was observed between megaspore mother cells and surrounding nucellus cells in principal component analysis of transcriptomic data .

What considerations should be made when using AT1G66250 antibody in co-immunoprecipitation experiments?

When designing co-immunoprecipitation (Co-IP) experiments to identify protein interaction partners of AT1G66250, researchers should consider:

• Antibody binding characteristics: The AT1G66250 antibody is a polyclonal antibody raised in rabbit against recombinant Arabidopsis thaliana AT1G66250 protein . This polyclonal nature provides good avidity but may increase background in Co-IP experiments.

• Optimization protocol:

  • Test different lysis buffers to identify optimal conditions for maintaining protein interactions

  • Consider crosslinking approaches (e.g., DSP or formaldehyde) to stabilize transient interactions

  • Test various antibody concentrations (typically 2-5 μg per mg of total protein)

  • Include appropriate controls (pre-immune serum, IgG from same species)

• Recommended Co-IP workflow:

  • Extract proteins in non-denaturing buffer with protease inhibitors

  • Pre-clear lysate with Protein A/G beads

  • Incubate cleared lysate with AT1G66250 antibody overnight at 4°C

  • Add Protein A beads (appropriate for rabbit IgG) and incubate 2-4 hours

  • Wash extensively to remove non-specific interactions

  • Elute and analyze by mass spectrometry or Western blotting

This approach can help identify novel interaction partners of AT1G66250, potentially revealing additional components of the plasmodesmata regulatory machinery in plant cells.

What are common issues encountered with AT1G66250 antibody and how can they be resolved?

Researchers working with AT1G66250 antibody may encounter several challenges. The following table outlines common issues and their solutions:

Importantly, when troubleshooting experiments with AT1G66250 antibody, remember that this protein is associated with plasmodesmata membranes, which may require specialized extraction and detection methods compared to soluble proteins.

How can researchers validate the specificity of AT1G66250 antibody in their experimental system?

Validating antibody specificity is crucial for reliable experimental results. For AT1G66250 antibody, consider these validation approaches:

• Genetic validation:

  • Test antibody reactivity in AT1G66250 knockout/knockdown lines (T-DNA insertion lines or CRISPR-edited plants)

  • Compare staining patterns between wild-type and mutant tissues

  • Ideal outcome: Reduced or absent signal in mutant tissues

• Biochemical validation:

  • Perform peptide competition assays using the immunizing peptide

  • Pre-incubate antibody with excess recombinant AT1G66250 protein before immunodetection

  • Ideal outcome: Significantly reduced signal after competition

• Orthogonal method validation:

  • Compare protein expression with mRNA expression data

  • Correlate antibody staining with fluorescent protein fusion localization

  • Use mass spectrometry to confirm identity of immunoprecipitated proteins

• Cross-species validation:

  • Test reactivity across species with known sequence conservation

  • Compare with expected cross-reactivity (Brassica napus, Brassica rapa, Populus trichocarpa, etc.)

  • Sequence alignment analysis to predict potential cross-reactivity

Thorough validation ensures that experimental observations truly reflect AT1G66250 biology rather than antibody artifacts or non-specific interactions.

How should researchers analyze co-localization data involving AT1G66250 and other plasmodesmata markers?

Co-localization studies examining the relationship between AT1G66250 and other plasmodesmata components require rigorous quantitative analysis:

• Image acquisition considerations:

  • Use confocal microscopy with appropriate controls for spectral bleed-through

  • Maintain consistent exposure settings across samples

  • Capture Z-stacks to account for the three-dimensional nature of plasmodesmata

• Quantitative co-localization analysis:

  • Calculate Pearson's correlation coefficient (values from -1 to +1)

  • Determine Manders' overlap coefficients (M1 and M2)

  • Use intensity correlation analysis (ICA) for more detailed relationship assessment

• Recommended workflow:

  • Process images with minimal manipulations (background subtraction only)

  • Define regions of interest (ROIs) around cell boundaries/plasmodesmata

  • Apply co-localization algorithms using ImageJ/Fiji with JACoP plugin

  • Set thresholds systematically using objective methods (e.g., Costes method)

  • Report both visual overlays and quantitative metrics

• Interpretation guidelines:

  • Pearson's coefficient > 0.5 suggests meaningful co-localization

  • Consider biological context when interpreting partial co-localization

  • Compare with known plasmodesmata markers (e.g., PDLP1, PDCB1)

  • Evaluate changes in co-localization across developmental stages or treatments

This approach provides robust evidence for the spatial relationship between AT1G66250 and other proteins within the plasmodesmata complex.

How might AT1G66250 antibody contribute to understanding beta-glucan dynamics during plant development?

The AT1G66250 antibody offers significant potential for advancing our understanding of beta-glucan regulation during plant development. Future research directions could include:

• Developmental time-course studies: Track AT1G66250 protein localization and abundance throughout ovule development to correlate with callose deposition patterns and symplastic connectivity changes.

• Response to environmental stresses: Investigate how AT1G66250 localization and activity change in response to abiotic stresses known to affect plasmodesmata function, such as pathogen attack, drought, or temperature fluctuations.

• Integration with emerging technologies:

  • Combine with super-resolution microscopy (STED, STORM) to examine nanoscale organization within plasmodesmata

  • Implement in proximity labeling approaches (BioID, APEX) to identify the protein neighborhood of AT1G66250

  • Utilize in single-cell proteomics to complement transcriptomic data on cell-type specific expression

• Functional studies: Use the antibody to assess changes in protein localization and abundance in plants with altered beta-glucan metabolism or plasmodesmata function, potentially revealing regulatory mechanisms.

These approaches could significantly advance our understanding of how beta-glucan dynamics contribute to developmental processes, particularly in reproductive tissues where recent research has highlighted the importance of symplastic regulation .

What role might AT1G66250 play in plant immune responses, and how can the antibody help investigate this?

While AT1G66250 has been primarily studied in developmental contexts, beta-1,3-glucanases often play important roles in plant immune responses. Future research could explore:

• Pathogen-induced changes: Monitor AT1G66250 localization and abundance following pathogen challenge, particularly with fungi containing beta-glucan cell walls.

• Callose-mediated defense responses: Investigate whether AT1G66250 contributes to the regulation of pathogen-induced callose deposition at cell walls and plasmodesmata.

• Signaling pathway integration: Examine how plant defense signaling pathways (salicylic acid, jasmonic acid) affect AT1G66250 expression and localization.

• Experimental approaches:

  • Immunolocalization following pathogen challenge

  • Western blot analysis of protein abundance in infected vs. healthy tissues

  • Co-immunoprecipitation to identify pathogen-induced protein interactions

  • Combine with genetic approaches in defense signaling mutants

This research direction could potentially reveal dual functions of AT1G66250 in both developmental regulation and pathogen defense, similar to what has been observed for other cell wall-modifying enzymes in plants.