At3g29070 Antibody

What is At3g29070 and why is it important in plant research?

At3g29070 is the gene identifier for Actin-7 in Arabidopsis thaliana, a key cytoskeletal protein essential for numerous developmental processes. Actin-7 has been identified across animals, plants, and protists but has specific functions in plant development. This protein plays critical roles in auxin-induced cellular changes, including cell division, expansion, differentiation, and organ initiation . The ACT7 gene product is rapidly and strongly induced in response to exogenous auxin, making it important for studying hormone-responsive pathways in plants . Additionally, Actin-7 is required for callus tissue formation, germination, and root growth, and is highly expressed in rapidly developing tissues . Its responsiveness to external stimuli, particularly hormones, makes At3g29070 antibodies valuable tools for researchers investigating plant development and stress responses.

How do I select the appropriate anti-At3g29070 (Actin-7) antibody clone for my research?

Selection of the optimal antibody clone for At3g29070 research requires consideration of your specific experimental goals and techniques. Based on available antibody resources, multiple monoclonal antibodies have been developed against Arabidopsis thaliana Actin-7, including clones 29G12.G5.G6, 33E8.C11.F5.D1, and 36H8.C12.H10.B6 . For first-time experiments, it is advisable to use all three monoclonal antibodies to determine which performs best for your specific experimental setup .

When selecting an antibody clone, consider:

• Technical application compatibility (WB, ELISA, IF)

• Cross-reactivity with actin isoforms from other species

• Epitope location and accessibility in your experimental conditions

• Published validation data for your specific application

These antibodies may complement experiments utilizing universal anti-actin antibodies (such as mAbGEa) when more specificity for Actin-7 is required .

What are the validated applications for At3g29070 antibodies in plant research?

At3g29070 antibodies have been validated for multiple molecular and cellular applications in plant research. Based on technical specifications, the following applications have been confirmed:

These antibodies are particularly useful in experiments investigating cytoskeletal reorganization during hormone responses, developmental transitions, and stress adaptation . For optimal results, preliminary titration experiments are recommended to determine the ideal concentration for your specific plant material and experimental conditions.

How can I use At3g29070 antibodies to study plant hormone responses?

At3g29070 (Actin-7) antibodies provide valuable tools for studying plant hormone responses, particularly auxin signaling. Since the ACT7 promoter and protein are rapidly and strongly induced by exogenous auxin , these antibodies can be used to:

• Visualize cytoskeletal reorganization during auxin-induced morphological changes using immunofluorescence microscopy

• Quantify Actin-7 protein expression levels after hormone treatment via Western blotting or ELISA

• Track tissue-specific expression patterns during hormone-induced developmental transitions

For experimental design, consider:

• Including appropriate hormone concentration gradients (10^-9 to 10^-5 M auxin)

• Time-course experiments (15 min to 24 hours post-treatment)

• Comparison with other hormone treatments to assess specificity

• Co-labeling with other cytoskeletal or signaling components

This approach enables researchers to correlate cytoskeletal dynamics with hormone-induced developmental changes at both cellular and tissue levels.

How can At3g29070 antibodies be incorporated into antibody-cell conjugation (ACC) techniques for enhanced plant cell studies?

Advanced researchers can leverage antibody-cell conjugation (ACC) technology to create novel experimental systems with At3g29070 antibodies. ACC allows antibodies to be directly coupled to cell surfaces without genetic modification, providing new approaches for studying plant cytoskeletal dynamics . For At3g29070 studies, consider these ACC methodologies:

• Metabolic sugar engineering with bioorthogonal reactions: Introduce azide moieties onto plant cell surfaces, then couple with DBCO-modified At3g29070 antibodies through click chemistry . This enables visualization of antibody localization while maintaining cell viability.

• Chemoenzymatic coupling using fucosyltransferase: Utilize H. pylori-derived α-1,3-fucosyltransferase to transfer At3g29070 antibodies coupled to GDP-fucose directly onto plant cell surface glycans . This one-step operation provides rapid coupling without genetic modification.

• DNA-hybridization based coupling: Attach single-stranded DNA to At3g29070 antibodies and complementary DNA to plant cell surface proteins, then join through DNA hybridization . This approach allows controlled, reversible antibody attachment.

These techniques enable novel applications such as tracking dynamic cytoskeletal reorganization during division and development, creating artificial cell-cell interactions, or developing new biosensors for plant hormone responses .

What strategies can resolve cross-reactivity issues when using At3g29070 antibodies in non-model plant species?

Cross-reactivity issues with At3g29070 antibodies in non-model plant species present significant challenges for comparative studies. While these antibodies were initially generated against Arabidopsis thaliana Actin-7, they may display reactivity toward multiple other species . To resolve cross-reactivity challenges:

• Epitope mapping and sequence alignment: Compare the Actin-7 sequences across your species of interest to identify regions of homology and divergence. Focus on epitope regions recognized by each antibody clone.

• Graduated validation protocol:

  • Begin with Western blot analysis using titrated antibody concentrations

  • Include multiple tissue types and developmental stages

  • Perform peptide competition assays to confirm specificity

  • Use siRNA/CRISPR knockdown controls in transformable species

• Multi-antibody approach: Utilize all three available monoclonal antibodies (29G12.G5.G6, 33E8.C11.F5.D1, 36H8.C12.H10.B6) alongside universal anti-actin antibodies to create a specificity profile .

• Mass spectrometry validation: Confirm antibody targets in immunoprecipitation experiments through proteomics analysis to identify all proteins being recognized in your non-model system.

This systematic approach allows researchers to determine which antibody clone provides optimal specificity for each non-model species, enabling more reliable comparative studies.

How should researchers interpret contradictory results between At3g29070 antibody labeling and gene expression data?

Discrepancies between At3g29070 antibody labeling and gene expression data are not uncommon and require careful analysis. Several biological and technical factors may contribute to these contradictions:

• Post-transcriptional regulation: Actin-7 protein levels may be regulated independently of mRNA abundance through:

  • Differential translation efficiency

  • Protein stability and turnover rates

  • Subcellular localization changes affecting antibody accessibility

• Antibody sensitivity thresholds: Immunodetection may have different sensitivity limits compared to transcript quantification methods.

• Isoform specificity challenges: At3g29070 antibodies may detect multiple actin isoforms despite being raised against Actin-7, particularly in complex samples .

To resolve these contradictions, implement:

• Parallel protein and mRNA quantification from the same samples

• Pulse-chase experiments to determine protein half-life

• Subcellular fractionation before analysis

• Complementary approaches such as fluorescent protein fusions

• Quantitative immunoprecipitation followed by mass spectrometry

In particular, researchers should consider that Actin-7 is responsive to external stimuli such as hormones , which may create temporal disconnects between transcription and protein accumulation during signal transduction events.

What quantitative analysis methods are most appropriate for At3g29070 immunofluorescence data in developing tissues?

Quantitative analysis of At3g29070 immunofluorescence data in developing tissues requires specialized approaches to account for the dynamic nature of cytoskeletal structures. For robust quantification:

• Filament organization metrics:

  • Measure filament length, thickness, and angular distribution

  • Calculate bundling factor (ratio of bundled to total filament signal)

  • Determine mesh size and density using network analysis algorithms

• Colocalization analysis with developmental markers:

  • Employ Pearson's or Manders' coefficients for spatial correlation

  • Use intensity correlation quotient (ICQ) for relationship strength

  • Implement object-based colocalization for discrete structures

• Temporal analysis in developing tissues:

  • Track filament dynamics through time-lapse imaging

  • Measure remodeling rates in response to developmental cues

  • Quantify polarization indices during asymmetric growth

• 3D reconstruction considerations:

  • Implement deconvolution to improve signal-to-noise ratio

  • Use appropriate z-step sizing for full volumetric reconstruction

  • Apply anisotropic corrections for elongated cells

These quantitative approaches are particularly relevant since Actin-7 is expressed in rapidly developing tissues and responds to external stimuli such as hormones , creating complex spatiotemporal patterns that require sophisticated analysis methods.

How can At3g29070 antibodies be used to study the relationship between auxin signaling and cytoskeletal dynamics?

At3g29070 antibodies provide powerful tools for investigating the mechanistic links between auxin signaling and cytoskeletal remodeling. Since ACT7 is rapidly and strongly induced by exogenous auxin , researchers can design experiments to visualize this relationship:

• Dual labeling approaches:

  • Co-immunolabeling of Actin-7 with auxin signaling components (TIR1/AFB receptors, Aux/IAA proteins)

  • Combined visualization of auxin reporters (DR5) with Actin-7 immunofluorescence

  • Triple labeling with microtubule markers to assess cytoskeletal crosstalk

• Pharmacological manipulation protocols:

  • Treatment with auxin transport inhibitors (NPA, TIBA) followed by Actin-7 immunolabeling

  • Application of auxin analogs with differential signaling properties

  • Cytoskeletal drug treatments (LatB, cytochalasin D) with auxin response monitoring

• Genetic background considerations:

  • Analysis in auxin signaling mutants (tir1, axr1, pin1)

  • Comparison with actin mutants to assess feedback mechanisms

  • Utilization of inducible expression systems for temporal control

Since Actin-7 is essential for germination and root growth , these approaches are particularly valuable for studying developmental transitions where auxin gradients direct growth responses through cytoskeletal reorganization.

What are the most effective protocols for using At3g29070 antibodies in chromatin immunoprecipitation experiments?

While not traditionally used for chromatin interactions, At3g29070 antibodies can be adapted for specialized chromatin immunoprecipitation (ChIP) protocols to investigate potential nuclear functions of Actin-7. Recent research has revealed non-canonical roles for actin in nuclear processes, including chromatin remodeling and transcriptional regulation. For ChIP applications with At3g29070 antibodies:

• Optimized nuclear isolation:

  • Use gentle extraction buffers containing cytoskeleton stabilizing agents

  • Implement density gradient centrifugation for pure nuclear fractions

  • Verify nuclear actin presence before proceeding to ChIP

• Modified crosslinking protocol:

  • Test graduated formaldehyde concentrations (0.1-3%)

  • Consider dual crosslinking with DSP or EGS for protein-protein stabilization

  • Optimize crosslinking time to preserve transient nuclear actin interactions

• Sonication parameters:

  • Implement milder sonication conditions to preserve protein-chromatin complexes

  • Use controlled enzymatic digestion as an alternative approach

  • Verify fragment size distribution before immunoprecipitation

• Validation controls:

  • Include cytoplasmic marker controls to assess contamination

  • Perform parallel IPs with multiple Actin-7 antibody clones (29G12.G5.G6, 33E8.C11.F5.D1, 36H8.C12.H10.B6)

  • Include IgG and input controls for normalization

This approach enables investigation of potential direct or indirect roles of Actin-7 in transcriptional regulation, particularly in auxin-responsive gene expression contexts.