At3g52030 Antibody

What is At3g52030 and what protein does it encode?

At3g52030 is the gene identifier for Actin-7 in Arabidopsis thaliana, a member of the actin gene family that encodes a critical cytoskeletal protein. Actin-7 is one of several actin isoforms found in A. thaliana, with distinctive expression patterns and regulatory mechanisms compared to other plant actins. The protein has been identified across plants, animals, and protists, highlighting its evolutionary conservation . Unlike constitutively expressed actin isoforms, Actin-7 shows distinctive developmental and stimulus-responsive expression patterns, making it an important target for developmental biology research.

What is the functional significance of Actin-7 in plant research?

Actin-7 serves multiple crucial functions in plant development and physiology:

• Essential for callus tissue formation during plant regeneration and tissue culture

• Required for normal germination processes and root growth

• Highly expressed in rapidly developing tissues

• Responsive to external stimuli, particularly phytohormones like auxin

• Involved in auxin-induced cellular responses including cell division, expansion, and differentiation

Unlike other actin isoforms, ACT7 demonstrates rapid and strong induction in response to exogenous auxin, suggesting its specialized role in hormone-mediated developmental processes. This makes Actin-7 antibodies particularly valuable for studying hormone signaling pathways and dynamic cytoskeletal rearrangements during plant development.

How does Actin-7 differ from other actin isoforms in experimental detection?

Actin-7 differs from other actin isoforms primarily in its:

These differences make Actin-7 antibodies valuable tools for distinguishing this isoform from other actins when studying tissue-specific and hormone-regulated developmental processes. Monoclonal antibodies against A. thaliana Actin-7 (such as clones 29G12.G5.G6, 33E8.C11.F5.D1, and 36H8.C12.H10.B6) have been developed specifically to recognize this protein .

How can I troubleshoot non-specific binding when using At3g52030 antibodies?

Non-specific binding is a common challenge when working with antibodies against highly conserved proteins like actins. For At3g52030 antibodies, consider these troubleshooting approaches:

• Antibody validation: Verify your antibody recognizes Actin-7 specifically by testing against knockout/knockdown lines or using peptide competition assays

• Blocking optimization: Test different blocking agents at various concentrations:

  • 5% non-fat dry milk in TBST (standard)

  • 3-5% BSA (often superior for phospho-specific antibodies)

  • Commercial plant-specific blocking reagents that reduce background

• Cross-reactivity management: If detecting cross-reactivity with other actin isoforms:

  • Increase antibody dilution (1:5000 to 1:10000 for Western blots)

  • Reduce primary antibody incubation time

  • Perform pre-adsorption against recombinant protein containing conserved actin regions

• Tissue-specific considerations: Cross-reactivity varies between tissue types due to differential actin isoform expression:

Implementing these strategies systematically can significantly reduce non-specific binding and improve experimental outcomes when working with At3g52030 antibodies .

What strategies exist for quantifying Actin-7 dynamics during auxin response?

Studying Actin-7 dynamics in response to auxin requires specialized approaches:

• Time-course immunoblotting: For quantifying protein level changes

  • Collect tissue samples at multiple timepoints after auxin treatment (5 min, 15 min, 30 min, 1h, 3h, 6h)

  • Process all samples simultaneously using standardized extraction and immunoblotting protocols

  • Include loading controls (non-auxin responsive proteins) for normalization

  • Use chemiluminescence detection with standard curves for quantification

• Immunolocalization analysis: For detecting subcellular redistribution

  • Fix tissues at precisely timed intervals after auxin treatment

  • Use gentle fixation methods to preserve dynamic actin structures

  • Perform dual labeling with organelle markers to track association changes

  • Employ confocal microscopy with Z-stack imaging for 3D visualization

• Live-cell imaging: For real-time dynamics (requires fusion proteins)

  • Generate Actin-7-GFP constructs under native promoter

  • Validate constructs against antibody staining patterns

  • Perform time-lapse imaging during auxin treatment

  • Use photobleaching techniques (FRAP) to measure turnover rates

• Analysis software recommendations:

  • ImageJ with FilamentTracker plugin for filament dynamics

  • CellProfiler for automated quantification across multiple samples

  • R with specialized cytoskeletal analysis packages for statistical comparison

These approaches, particularly when combined, provide comprehensive quantitative data on both expression level changes and subcellular reorganization of Actin-7 during auxin response .

How can I validate the specificity of At3g52030 antibodies in my experimental system?

Rigorous validation is essential when working with antibodies against conserved proteins like Actin-7. Implement these validation strategies:

• Genetic validation:

  • Test antibody against Actin-7 knockout/knockdown lines (expecting reduced/absent signal)

  • Compare staining patterns in overexpression lines (expecting increased signal intensity)

  • Examine tissue-specific expression patterns that should match known ACT7 expression domains

• Biochemical validation:

  • Perform peptide competition assays using synthetic peptides from unique regions of Actin-7

  • Conduct immunoprecipitation followed by mass spectrometry to confirm target identity

  • Test cross-reactivity against recombinant Actin-1 through Actin-11 proteins

• Orthogonal validation:

  • Compare antibody results with RNA expression data (qRT-PCR, RNA-seq)

  • Validate against fluorescent protein fusion constructs of Actin-7

  • Compare results across different antibody clones targeting different epitopes

• Validation data reporting:

  • Document complete validation data in laboratory notebooks and publications

  • Include positive and negative controls in all experimental figures

  • Report antibody catalog numbers, clone designations, and dilutions used

Thorough validation ensures experimental reproducibility and reliable interpretation of results when using At3g52030 antibodies .

What are the optimal fixation conditions for immunolocalization of Actin-7 in plant tissues?

Optimizing fixation is critical for preserving Actin-7 structures while maintaining antibody epitope accessibility:

• Paraformaldehyde fixation (recommended for most applications):

  • Prepare fresh 4% paraformaldehyde in PBS (pH 7.2)

  • Add 0.1% Triton X-100 to improve penetration

  • Fix tissues for 60-90 minutes at room temperature

  • Wash 3x15 minutes in PBS to remove fixative completely

• Methanol fixation (alternative for certain applications):

  • Pre-chill 100% methanol to -20°C

  • Immerse samples for exactly 10 minutes

  • Transfer directly to PBS at room temperature

  • This method often provides better preservation of certain actin structures but may reduce epitope accessibility

• Tissue-specific optimization:

• Post-fixation processing:

  • Cell wall digestion with 1% cellulase, 0.5% macerozyme (20-30 min)

  • Permeabilization with 0.5% Triton X-100 (15 min)

  • Blocking with 3% BSA in PBS (2 hours)

  • Antibody dilution: 1:200-1:500 for primary (overnight, 4°C)

These optimized conditions preserve the native organization of the actin cytoskeleton while allowing specific detection of Actin-7 by antibodies .

What protocol modifications are needed for detecting At3g52030 protein in different experimental contexts?

Different experimental approaches require specific protocol modifications:

• Western blot optimization:

  • Sample preparation: Add phosphatase inhibitors to preserve modification states

  • Gel percentage: 12% acrylamide gels provide optimal resolution for 42kDa Actin-7

  • Transfer conditions: Semi-dry transfer at 15V for 30 minutes works well

  • Blocking: 5% milk in TBST (1 hour, room temperature)

  • Primary antibody: 1:2000-1:5000 dilution (overnight, 4°C)

  • Detection: HRP-conjugated secondary with standard ECL substrates

• Immunoprecipitation protocol:

  • Lysis buffer: 50mM Tris-HCl pH 7.5, 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1mM EDTA, protease inhibitors

  • Pre-clearing: 1 hour with Protein A/G beads to reduce background

  • Antibody binding: 5μg antibody per 500μg total protein (overnight, 4°C)

  • Wash conditions: 4x with lysis buffer, 2x with PBS

  • Elution: Either with SDS sample buffer (denaturing) or peptide competition (native)

• Immunofluorescence optimization:

  • Antigen retrieval: 10mM sodium citrate, pH 6.0, 95°C, 10 minutes

  • Signal amplification: TSA (tyramide signal amplification) for low-abundance detection

  • Counterstains: DAPI for nuclei, selected organelle markers for co-localization

  • Mounting media: Anti-fade reagent with pH stabilizers improves signal longevity

• Flow cytometry applications:

  • Cell preparation: Gentle protoplasting using reduced enzyme concentrations

  • Fixation: 2% PFA, 15 minutes, on ice

  • Permeabilization: 0.1% saponin rather than harsher detergents

  • Antibody dilution: 1:100-1:200 (higher than for other applications)

  • Controls: Include isotype control and secondary-only samples

These protocol modifications significantly improve detection sensitivity and specificity across different experimental contexts .

How can I effectively use At3g52030 antibodies to study actin dynamics during auxin response?

Investigating the dynamic relationship between Actin-7 and auxin signaling requires specialized experimental approaches:

• Controls and validation:

  • Include cytoskeleton-disrupting drugs (Latrunculin B, Cytochalasin D) as positive controls

  • Test auxin transport inhibitors (NPA, TIBA) to confirm specificity

  • Validate findings in auxin signaling mutants (tir1, arf7/19)

  • Compare responses of multiple actin isoforms using specific antibodies

This integrated approach provides comprehensive insights into the role of Actin-7 in mediating auxin-induced cellular responses, particularly in rapidly developing tissues and germination processes .

What experimental design best demonstrates the relationship between Actin-7 and callus formation?

Given Actin-7's essential role in callus formation, these experimental approaches can elucidate the underlying mechanisms:

• Comparative analysis protocol:

  • Induce callus formation on wild-type and act7 mutant explants

  • Compare callus induction efficiency, growth rates, and morphology

  • Sample tissues at defined intervals during callus development (0, 3, 7, 14, 21 days)

  • Process for both protein analysis and immunohistochemistry

• Protein dynamics analysis:

  • Extract proteins from developing callus at multiple timepoints

  • Quantify Actin-7 protein levels via western blotting

  • Compare with other actin isoforms using specific antibodies

  • Correlate protein levels with callus development stages

• Cytoskeletal architecture study:

  • Perform immunofluorescence on callus sections at different developmental stages

  • Image using high-resolution confocal microscopy

  • Analyze changes in actin filament organization during dedifferentiation

  • Compare with other cytoskeletal elements (microtubules, intermediate filaments)

• Rescue experiment design:

  • Transform act7 mutants with Actin-7 under native or inducible promoters

  • Assess restoration of callus formation capacity

  • Use fluorescently tagged constructs to visualize dynamics in vivo

  • Test structure-function relationships using point mutations

• Data collection parameters:

These experimental approaches provide mechanistic insights into how Actin-7 contributes to the cellular reorganization required for callus formation, with implications for plant regeneration and tissue culture techniques .

How can At3g52030 antibodies be combined with emerging technologies for cytoskeletal research?

Recent technological advances offer new opportunities for Actin-7 research:

• Super-resolution microscopy applications:

  • STORM/PALM imaging allows visualization of individual actin filaments below the diffraction limit

  • SIM provides 2x resolution improvement with standard immunofluorescence protocols

  • Expansion microscopy physically enlarges samples for improved resolution with standard confocal equipment

  • Recommended antibody dilutions: 1:100-1:200 for STORM, 1:200-1:500 for SIM

• Live-cell imaging integration:

  • Combine fixed-cell antibody imaging with live-cell GFP-tagged Actin-7 imaging

  • Correlative Light and Electron Microscopy (CLEM) to connect ultrastructure with specific antibody labeling

  • Optimize fixation timing to capture transient cytoskeletal states observed in live imaging

  • Use fiduciary markers for precise alignment between live and fixed images

• Proximity labeling approaches:

  • TurboID or APEX2 fusions to Actin-7 to identify proximal interacting proteins

  • Validate interactions using co-immunoprecipitation with At3g52030 antibodies

  • Map interaction networks during developmental transitions or hormone responses

  • Correlate with protein-protein interaction predictions from structural models

• Antibody-based biosensors:

  • Develop FRET-based sensors using antibody-derived binding domains

  • Create split-GFP complementation systems for detecting Actin-7 interactions

  • Design antibody-based optogenetic tools for manipulating actin dynamics

  • Validate sensor specificity using conventional At3g52030 antibodies

These emerging technologies, when combined with well-validated At3g52030 antibodies, enable unprecedented insights into Actin-7 dynamics and functions in plant development and hormone responses .

What are the most promising antibody-based approaches for studying Actin-7 in developmental contexts?

Several innovative approaches show particular promise for developmental studies:

• Tissue clearing with antibody penetration:

  • Clear plant tissues using ClearSee or PEA-CLARITY protocols

  • Perform whole-mount immunolabeling with At3g52030 antibodies

  • Image entire organs or seedlings with light-sheet microscopy

  • Reconstruct 3D models of Actin-7 distribution throughout development

• Single-cell analysis integration:

  • Combine antibody-based flow cytometry with single-cell RNA-seq

  • Sort cells based on Actin-7 levels or organization patterns

  • Profile transcriptomes of populations with distinct cytoskeletal states

  • Correlate cytoskeletal organization with developmental gene expression programs

• Tissue-specific perturbation analysis:

  • Express nanobodies derived from At3g52030 antibodies in specific tissues

  • Disrupt Actin-7 function in a spatially controlled manner

  • Monitor developmental consequences of tissue-specific disruption

  • Compare with traditional genetic approaches using tissue-specific promoters

• Comparative developmental studies across species:

  • Test At3g52030 antibody cross-reactivity with other plant species

  • Compare Actin-7 expression and organization patterns across evolutionary distance

  • Identify conserved and divergent aspects of actin regulation in development

  • Create evolutionary models of cytoskeletal function in plant morphogenesis

These approaches leverage the specificity of At3g52030 antibodies to address fundamental questions about the role of the actin cytoskeleton in plant development, with particular relevance to understanding auxin-regulated growth processes and cellular differentiation mechanisms .

What resources are available for researchers working with At3g52030 antibodies?

Researchers have access to several important resources:

• Commercial antibody sources:

  • Multiple validated monoclonal antibodies are available (clones 29G12.G5.G6, 33E8.C11.F5.D1, 36H8.C12.H10.B6)

  • These recognize Arabidopsis thaliana Actin-7 specifically

  • May display reactivity toward homologous proteins in other plant species

• Research communities and shared protocols:

  • Arabidopsis Biological Resource Center maintains relevant genetic resources

  • Plant cytoskeleton research networks share optimized protocols

  • Specialized imaging facilities often have experience with plant actin visualization

• Genetic resources:

  • Multiple act7 mutant alleles available through stock centers

  • Fluorescent protein fusion lines for comparative studies

  • Inducible expression systems for structure-function analysis

• Bioinformatic tools:

  • Sequence alignment tools for comparing actin isoforms

  • Epitope prediction software for antibody characterization

  • Image analysis packages optimized for cytoskeletal quantification

These resources collectively support diverse research applications of At3g52030 antibodies in plant biology, cell biology, and developmental research contexts .

How can researchers design controlled comparative studies of actin isoforms using specific antibodies?

Designing rigorous comparative studies requires careful planning:

• Experimental design considerations:

  • Include multiple actin isoform antibodies in parallel experiments

  • Process all samples simultaneously to minimize technical variation

  • Include appropriate genetic controls (knockout/knockdown lines)

  • Design time-courses that capture relevant developmental transitions

• Controls and validations:

  • Validate each antibody's specificity against its target isoform

  • Perform western blots on multiple tissue types to confirm isoform-specific detection

  • Include isoform-specific peptide competition controls

  • Test cross-reactivity systematically against all known actin isoforms

• Data analysis approach:

  • Normalize signal intensities appropriately for fair comparisons

  • Use ratiometric analysis between different actin isoforms

  • Apply appropriate statistical tests for comparing expression patterns

  • Create visualization methods that highlight differences in spatial/temporal patterns

• Recommended experimental matrix: