At3g17155 Antibody

What is At3g17155 and why are antibodies against it important for plant research?

At3g17155 is the gene identifier for Actin-7 in Arabidopsis thaliana, a critical component of the plant cytoskeleton. Antibodies targeting this protein are essential tools for studying cytoskeletal dynamics in plant development and stress responses. Actin-7 has been identified across multiple kingdoms including animals, plants, and protists, but plays particularly important roles in plant development. The protein is rapidly and strongly induced in response to the phytohormone auxin, which triggers various developmental changes including cell division, expansion, differentiation, and organ initiation . Using specific antibodies allows researchers to track the expression, localization, and interactions of Actin-7 during these fundamental plant processes.

What are the primary biological functions of the At3g17155/Actin-7 protein?

Actin-7 serves multiple critical functions in plant biology:

• Essential role in callus tissue formation

• Required for proper germination and root growth

• Expressed predominantly in rapidly developing tissues

• Responsive to external stimuli, particularly plant hormones

• Participates in auxin-mediated developmental responses

• Contributes to cytoskeletal reorganization during various growth phases

The actin cytoskeleton is believed to play an active role in mediating cellular responses to hormones like auxin by directing specific changes in cell morphology and cytoarchitecture. Actin-7 specifically has been identified as necessary for these fundamental processes, making it a valuable target for developmental biology research .

What types of antibodies are available for detecting At3g17155/Actin-7?

Currently available antibodies for At3g17155/Actin-7 include:

These mouse monoclonal antibodies were specifically generated against Arabidopsis thaliana Actin-7 and have been purified using Protein G. They are typically stored in PBS with 0.05% (w/v) sodium azide and should be kept at -20°C for optimal preservation . It is advisable for researchers to test all three monoclonal antibodies in initial qualitative experiments to determine which is most suitable for specific experimental conditions.

How should researchers optimize Western blot protocols for At3g17155/Actin-7 antibodies?

When conducting Western blot analyses with At3g17155 antibodies, researchers should consider several optimization factors:

• Sample preparation: Arabidopsis tissues should be flash-frozen and ground in liquid nitrogen before adding extraction buffer containing protease inhibitors to preserve protein integrity.

• Protein separation: Use 10-12% SDS-PAGE gels for optimal separation of Actin-7 (approximately 42 kDa).

• Antibody dilution testing: Begin with a 1:1000 dilution of the primary antibody and adjust based on signal strength.

• Specificity controls: Include negative controls (non-plant tissues) and positive controls (tissues known to express high levels of Actin-7, such as rapidly growing seedlings).

• Complementary approach: Consider using all three available monoclonal antibodies (29G12.G5.G6, 33E8.C11.F5.D1, 36H8.C12.H10.B6) in preliminary experiments to determine which provides the most specific signal for your particular sample type .

• Comparison with universal anti-actin antibodies: The At3g17155 antibodies may be used to complement experiments carried out with universal anti-actin antibodies (such as mAbGEa) to verify specificity to Actin-7 versus other actin isoforms .

What are the recommended immunofluorescence protocols for visualizing At3g17155/Actin-7 in plant tissues?

For successful immunofluorescence experiments with At3g17155 antibodies:

• Fixation: Fix fresh plant tissues in 4% paraformaldehyde in PBS for 1-2 hours, followed by permeabilization with 0.1-0.5% Triton X-100.

• Blocking: Block with 3-5% BSA in PBS for at least 1 hour to reduce non-specific binding.

• Primary antibody incubation: Incubate with the At3g17155 antibody (starting at 1:100 dilution) overnight at 4°C. Testing all three available monoclonal antibodies initially is recommended to determine optimal performance.

• Detection: Use appropriate fluorophore-conjugated secondary antibodies (anti-mouse IgG) and include DAPI for nuclear counterstaining.

• Controls: Include controls omitting primary antibody and test tissues known to have varied Actin-7 expression levels.

• Co-localization studies: Consider co-staining with other cytoskeletal markers to examine interactions between Actin-7 and other structural proteins.

• Confocal microscopy settings: Optimize pinhole, gain, and laser power settings for detecting specific signals while minimizing background fluorescence .

How can At3g17155 antibodies be used to study stress-induced cytoskeletal reorganization?

At3g17155/Actin-7 antibodies can be powerful tools for investigating cytoskeletal responses to various stresses:

• Experimental design: Compare Actin-7 distribution patterns in control plants versus plants exposed to specific stressors (heat, drought, DNA damage) at defined time intervals.

• Quantification methods: Implement digital image analysis to quantify changes in fluorescence intensity, filament organization, and co-localization with stress response proteins.

• Tissue-specific responses: Examine how different tissue types (root tips, leaf mesophyll, vascular tissues) exhibit varied Actin-7 reorganization under stress conditions.

• Temporal dynamics: Track the time course of cytoskeletal changes following stress application to identify early versus late responses.

• Integration with cell cycle analysis: Since Actin-7 plays roles in growth and development, correlate cytoskeletal changes with cell cycle progression markers during stress .

Research has shown that stress conditions like DNA damage and heat stress can trigger cell cycle arrest mechanisms in Arabidopsis, where proteins like ANAC044 and ANAC085 play crucial roles in G2 arrest . Antibodies against Actin-7 could help identify how cytoskeletal reorganization coordinates with these cell cycle regulatory mechanisms during stress responses.

What approaches can be used to study the relationship between At3g17155/Actin-7 and auxin signaling?

To investigate the interplay between Actin-7 and auxin signaling pathways:

• Time-course experiments: Track Actin-7 protein levels and localization at defined intervals following auxin treatment using both immunoblotting and immunofluorescence with At3g17155 antibodies.

• Genetic interaction studies: Compare Actin-7 expression and localization patterns in wild-type plants versus auxin signaling mutants.

• Pharmacological approaches: Combine auxin treatments with cytoskeletal inhibitors (cytochalasin D, latrunculin B) to determine the requirement of functional actin filaments for auxin responses.

• Co-immunoprecipitation: Use At3g17155 antibodies for co-IP experiments to identify auxin-dependent protein interactions with Actin-7.

• Promoter-reporter studies: Combine antibody-based protein detection with transcriptional reporter assays to correlate protein levels with gene expression changes.

Since research has established that the Actin-7 promoter and protein are rapidly and strongly induced in response to exogenous auxin , these approaches can help elucidate the molecular mechanisms connecting hormone signaling with cytoskeletal dynamics.

How can researchers investigate the role of At3g17155/Actin-7 in DNA damage response pathways?

For studying Actin-7's potential involvement in DNA damage responses:

• DNA damage induction: Treat plants with DNA-damaging agents like bleomycin, zeocin, or UV radiation at controlled doses.

• Cell cycle correlation: Use flow cytometry in combination with immunofluorescence to correlate Actin-7 distribution with cell cycle phases during DNA damage.

• Co-localization with DDR proteins: Examine potential co-localization of Actin-7 with known DNA damage response proteins such as SOG1, ANAC044, and ANAC085.

• Transgenic approaches: Compare Actin-7 antibody staining patterns in wild-type versus DNA damage response mutants (sog1, anac044, anac085).

• Chromatin association: Investigate whether Actin-7 associates with chromatin during DNA damage using chromatin immunoprecipitation followed by immunoblotting with At3g17155 antibodies.

Research has demonstrated that DNA damage triggers a complex signaling network in Arabidopsis involving transcription factors like SOG1, ANAC044, and ANAC085, which regulate cell cycle arrest at the G2 checkpoint . Since Actin-7 is involved in growth and development processes, investigating its potential role in these stress response pathways could reveal new insights into cytoskeletal functions during genomic stress.

How can researchers address non-specific binding when using At3g17155 antibodies?

Non-specific binding is a common challenge when working with plant antibodies. To improve specificity:

• Increase blocking stringency: Extend blocking time to 2-3 hours and consider alternative blocking agents like 5% non-fat dry milk or commercial blocking buffers specifically designed for plant tissues.

• Antibody titration: Perform careful dilution series (1:100 to 1:5000) to determine the optimal concentration that maximizes specific signal while minimizing background.

• Pre-absorption: Pre-absorb the antibody with non-target plant tissues or recombinant non-target actin isoforms to remove cross-reactive antibodies.

• Alternative detection methods: If one application (e.g., Western blot) shows non-specific binding, try alternative methods like ELISA or immunoprecipitation to confirm results.

• Multiple antibody validation: Compare results using all three available monoclonal antibodies (29G12.G5.G6, 33E8.C11.F5.D1, 36H8.C12.H10.B6) to identify which provides the highest specificity for your experimental system .

• Genetic controls: Include actin-7 knockout or knockdown plant samples as negative controls to confirm antibody specificity.

What are the key considerations for quantifying At3g17155/Actin-7 expression in different tissues?

For accurate quantification of Actin-7 across diverse plant tissues:

• Sample normalization: Standardize protein extraction methods and loading quantities across different tissue types, considering that protein extraction efficiency may vary by tissue.

• Reference proteins: Identify stable reference proteins appropriate for each tissue type being compared, as traditional housekeeping genes may vary in expression across tissues or conditions.

• Detection sensitivity: For tissues with low Actin-7 expression, enhance detection using more sensitive methods such as chemiluminescence for Western blots or signal amplification systems for immunofluorescence.

• Calibration curves: Generate standard curves using recombinant Actin-7 protein to enable absolute quantification.

• Technical replication: Perform multiple technical replicates and biological replicates to account for natural variation.

• Image analysis: Use appropriate software for densitometric analysis of Western blots or fluorescence intensity measurements in microscopy, applying consistent thresholding criteria across all samples.

• Statistical validation: Apply appropriate statistical tests to determine the significance of observed differences in Actin-7 levels between tissues or conditions.

How might At3g17155/Actin-7 antibodies be utilized in studying plant cell cycle regulation?

Actin-7 antibodies present valuable opportunities for investigating cell cycle-related cytoskeletal dynamics:

• Cell cycle synchronization: Combine cell cycle synchronization protocols with immunofluorescence to track Actin-7 distribution patterns at specific cell cycle phases.

• Co-immunoprecipitation with cell cycle regulators: Use At3g17155 antibodies for pull-down experiments to identify potential interactions with cell cycle proteins like cyclins, CDKs, or MYB transcription factors.

• G2 arrest studies: Investigate potential roles of Actin-7 in stress-induced G2 arrest, which has been shown to involve transcription factors like ANAC044 and ANAC085 in Arabidopsis .

• Cytokinesis focus: Examine Actin-7 localization during cell plate formation and cytokinesis in dividing plant cells.

• Combined cytoskeletal tracking: Implement dual-labeling approaches to simultaneously visualize Actin-7 and microtubules during cell cycle progression.

Research has established that cell cycle arrest mechanisms in plants involve complex regulatory networks, including NAC-type transcription factors and MYB transcription factors that control G2/M-specific genes . Investigating potential connections between Actin-7 dynamics and these regulatory pathways could reveal new insights into how the cytoskeleton integrates with cell cycle control.

What methodological approaches can be used to study post-translational modifications of At3g17155/Actin-7?

To investigate post-translational modifications (PTMs) of Actin-7:

• PTM-specific antibodies: Use antibodies specifically targeting common actin PTMs (phosphorylation, acetylation, ubiquitination) in combination with general At3g17155 antibodies.

• Two-dimensional gel electrophoresis: Separate Actin-7 isoforms based on both molecular weight and isoelectric point to detect charge-altering modifications.

• Mass spectrometry analysis: Immunoprecipitate Actin-7 using At3g17155 antibodies followed by mass spectrometry to identify and map specific modification sites.

• Treatment with modifying enzymes: Treat protein extracts with phosphatases, deacetylases, or deubiquitinases prior to immunoblotting to confirm the presence of specific modifications.

• Site-directed mutagenesis: Compare antibody reactivity between wild-type Actin-7 and variants with mutations at potential modification sites in transgenic plants.

• Stress condition comparisons: Examine how various stressors (hormones, heat, DNA damage) affect the PTM status of Actin-7 using modification-specific detection methods.

Understanding Actin-7's post-translational modifications could provide insight into how this cytoskeletal protein is regulated during development and stress responses, potentially connecting to broader regulatory networks like those involved in cell cycle control and stress adaptation.