At1g71900 Antibody

What is At1g71900 and what physiological roles does it play in plant development?

At1g71900 is the gene locus for Actin-7 in Arabidopsis thaliana, encoding a critical cytoskeletal protein involved in numerous developmental processes. ACT7 has been identified as essential for callus tissue formation, germination, and root growth, making it a vital component of plant development and morphogenesis. Unlike other actin isoforms, ACT7 is specifically expressed in rapidly developing tissues and shows unique responsiveness to external stimuli, particularly hormonal signals such as auxin .

The phytohormone auxin induces a wide array of changes during plant growth and development, including cell division, cell expansion, cell differentiation, and organ initiation. The actin cytoskeleton, particularly ACT7, plays an active role in facilitating these responses by directing specific changes in cell morphology and cytoarchitecture . Research has demonstrated that the promoter and protein product of ACT7 are rapidly and strongly induced in response to exogenous auxin, establishing its role in auxin-mediated developmental pathways.

When investigating At1g71900 function, researchers should consider its differential expression patterns across various tissues and developmental stages, as this contributes to its specialized roles in plant growth and adaptation.

What types of At1g71900 antibodies are available for research applications?

Several monoclonal antibodies against Arabidopsis thaliana Actin-7 have been developed for research purposes. From the available data, mouse monoclonal antibodies generated against A. thaliana Actin-7 include specific clones such as 29G12.G5.G6, 33E8.C11.F5.D1, and 36H8.C12.H10.B6 . These antibodies have been validated for various applications including Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence (IF).

When selecting an appropriate antibody, researchers should consider:

• The specific experimental application (WB, ELISA, IF, etc.)

• The level of specificity required for distinguishing ACT7 from other actin isoforms

• The detection method to be employed

For first-time qualitative experimental setups, it is advisable to use all three monoclonal antibodies to determine which is most suitable for a specific experiment. These can be used to complement experiments carried out with mAbGEa universal anti-actins antibody for comparative analyses .

What are the optimal protocols for At1g71900 antibody-based Western blotting?

Western blotting using At1g71900 antibodies requires specific optimization steps to ensure reliable detection of Actin-7 among the highly conserved actin family proteins. The following methodological approach is recommended:

• Sample preparation: Extract plant tissues in a buffer containing protease inhibitors to prevent degradation of actin proteins. Flash-freezing samples in liquid nitrogen before grinding is recommended to preserve protein integrity.

• Protein separation: Use 10-12% SDS-PAGE gels for optimal separation of actin proteins, which have a molecular weight of approximately 42 kDa.

• Transfer conditions: Transfer to PVDF membranes at 100V for 60-90 minutes in standard transfer buffer with 20% methanol.

• Blocking: Block membranes with 5% non-fat dry milk in TBST (TBS with 0.1% Tween-20) for 1 hour at room temperature.

• Primary antibody incubation: Dilute At1g71900 antibodies in blocking solution (recommended starting dilutions: 1:1000 to 1:2000) and incubate overnight at 4°C.

• Washing and secondary antibody: Wash membranes thoroughly with TBST before incubating with appropriate secondary antibodies (typically anti-mouse IgG) conjugated to HRP or fluorescent tags.

• Controls: Include both positive controls (known ACT7-expressing tissues) and negative controls to validate specificity.

To address the challenge of distinguishing ACT7 from other actin isoforms, researchers may need to perform additional validation steps, such as using ACT7 knockout mutants as negative controls or confirming results with multiple anti-ACT7 antibody clones.

How should At1g71900 antibodies be stored and handled for optimal performance?

Proper storage and handling of At1g71900 antibodies are crucial for maintaining their specificity and activity. Based on standard antibody storage protocols and available information, the following guidelines are recommended:

• Storage conditions: Store antibodies at -20°C in PBS buffer containing 0.05% (w/v) sodium azide as a preservative .

• Aliquoting: Upon receipt, divide the antibody into small single-use aliquots to avoid repeated freeze-thaw cycles which can damage antibody structure and reduce activity.

• Thawing process: Thaw aliquots slowly on ice or at 4°C rather than at room temperature to preserve antibody integrity.

• Working dilutions: Prepare working dilutions fresh on the day of use and keep at 4°C. Avoid storing diluted antibodies for extended periods.

• Shipping conditions: Antibodies are typically shipped with cold packs to maintain temperature stability during transport .

• Avoiding contamination: Use sterile technique when handling antibodies to prevent microbial contamination.

For long-term storage stability, purification methods such as Protein G have been employed for At1g71900 antibodies, which helps maintain their functional properties . Researchers should conduct regular quality control tests, such as activity assays, to ensure antibody performance has not deteriorated over time.

How can At1g71900 antibodies be optimized for immunofluorescence studies of plant cytoskeletal dynamics?

Immunofluorescence (IF) using At1g71900 antibodies presents unique challenges due to the plant cell wall, abundant cytoplasmic components, and the dynamic nature of the actin cytoskeleton. To achieve high-quality imaging of ACT7 dynamics, researchers should consider the following methodological optimizations:

• Fixation protocol: The choice between chemical fixatives (e.g., paraformaldehyde, glutaraldehyde) and physical methods affects actin preservation. A combination of 4% paraformaldehyde with 0.1% glutaraldehyde often provides good structural preservation of actin filaments.

• Cell wall digestion: Partial enzymatic digestion of cell walls with cellulase and pectinase improves antibody penetration. Optimize enzyme concentration and incubation time to maintain cellular integrity while allowing sufficient permeabilization.

• Permeabilization: After fixation, treat samples with 0.1-0.5% Triton X-100 to permeabilize membranes and enhance antibody access to intracellular antigens.

• Blocking strategy: Use 2-5% BSA or normal serum from the secondary antibody host species, supplemented with 0.1% Tween-20, to reduce non-specific binding.

• Antibody selection: Based on the available information, all three monoclonal antibodies (29G12.G5.G6, 33E8.C11.F5.D1, and 36H8.C12.H10.B6) have been validated for IF applications . Initial experiments should compare their performance to identify the optimal clone.

• Signal amplification: For low abundance or difficult-to-detect ACT7 populations, consider using tyramide signal amplification or highly sensitive detection systems.

• Multi-channel imaging: Co-staining with markers for other cellular structures (e.g., microtubules, organelles) provides contextual information about ACT7 localization and function.

Advanced research applications may include live-cell imaging of ACT7 dynamics by developing fusion proteins or nanobody-based detection systems, though these approaches require extensive validation against antibody-based methods to ensure consistency in ACT7 detection.

What strategies can be employed to study At1g71900 (ACT7) role in auxin-mediated developmental processes?

Investigating the relationship between ACT7 and auxin signaling requires sophisticated experimental approaches combining molecular, cellular, and physiological techniques. The following methodological strategies are recommended:

• Hormone response assays: Treat plant tissues with various concentrations of auxin (e.g., IAA, NAA, 2,4-D) and monitor ACT7 expression levels using At1g71900 antibodies via Western blotting or immunofluorescence. Time-course experiments can reveal the kinetics of ACT7 induction following auxin treatment .

• Promoter-reporter fusions: While antibody-based approaches detect the protein, complementary studies using ACT7 promoter fused to reporter genes (GUS, GFP) can reveal transcriptional regulation patterns in response to auxin.

• Genetic approaches: Compare ACT7 protein levels and localization patterns in wild-type plants versus auxin signaling mutants (e.g., tir1, axr1) using At1g71900 antibodies to establish regulatory relationships.

• Co-immunoprecipitation: Use At1g71900 antibodies to pull down ACT7 and associated proteins from auxin-treated and untreated samples to identify interaction partners that may mediate auxin responses.

• Pharmacological studies: Combine auxin treatments with actin-disrupting drugs (e.g., latrunculin B, cytochalasin D) and monitor developmental outcomes to dissect the specific contribution of ACT7 dynamics to auxin-mediated processes.

• Tissue-specific analyses: Apply At1g71900 antibodies to specific developmental contexts, such as:

  • Root growth and gravitropism assays

  • Callus induction and regeneration

  • Early embryogenesis

  • Organ initiation at shoot apical meristems

• Subcellular localization: Use high-resolution microscopy techniques (confocal, super-resolution) with At1g71900 antibodies to track changes in ACT7 filament organization following auxin treatment.

These approaches require careful experimental design with appropriate controls to distinguish ACT7-specific effects from general actin cytoskeletal responses to auxin.

How can the specificity of At1g71900 antibodies be validated in experimental setups?

Validating the specificity of At1g71900 antibodies is crucial for experimental reliability, especially given the high sequence similarity among actin isoforms. A comprehensive validation strategy should include:

• Genetic validation: Test antibody reactivity in act7 knockout or knockdown mutants compared to wild-type plants. Specific At1g71900 antibodies should show reduced or absent signal in act7 mutants.

• Peptide competition assays: Pre-incubate antibodies with purified ACT7 protein or immunizing peptide before application to samples. Specific binding should be blocked by the competing antigen, resulting in signal reduction.

• Western blot analysis: Beyond detecting the expected ~42 kDa band for ACT7, perform additional tests:

  • Compare banding patterns across different plant tissues with known differential expression of ACT7

  • Analyze multiple actin mutants to confirm isoform specificity

  • Perform 2D gel electrophoresis to separate actin isoforms based on pI differences before immunoblotting

• Mass spectrometry verification: Immunoprecipitate proteins using At1g71900 antibodies and identify the pulled-down proteins by mass spectrometry to confirm ACT7 enrichment.

• Cross-reactivity assessment: Test antibodies against recombinant versions of different actin isoforms to quantify relative binding affinities.

• Immunohistochemical pattern analysis: Compare immunolabeling patterns with known ACT7 expression domains from transcriptomic data or promoter-reporter studies.

• Multiple antibody verification: Compare results using different monoclonal antibody clones (29G12.G5.G6, 33E8.C11.F5.D1, and 36H8.C12.H10.B6) targeting different epitopes of ACT7 .

What approaches can be used to distinguish At1g71900 (ACT7) from other actin isoforms in Arabidopsis?

Distinguishing ACT7 from other actin isoforms presents a significant challenge due to the high sequence conservation among plant actins. Several methodological approaches can help achieve isoform-specific detection:

• Epitope selection: When possible, use antibodies raised against unique regions of ACT7 that differ from other actin isoforms. The variable N-terminal region often provides the greatest sequence divergence among actins.

• Combinatorial antibody approach: Use multiple At1g71900-specific antibody clones (29G12.G5.G6, 33E8.C11.F5.D1, and 36H8.C12.H10.B6) targeting different epitopes to increase detection specificity .

• Differential extraction protocols: Optimize extraction conditions that may preferentially isolate ACT7 based on potential differences in solubility, binding partners, or subcellular localization compared to other actins.

• Expression pattern analysis: Leverage the known expression patterns of ACT7, which is uniquely expressed in rapidly developing tissues and responds to hormonal stimuli such as auxin . Designing experiments around these distinctive expression contexts can help distinguish ACT7-specific signals.

• Genetic background strategy: Perform experiments in genetic backgrounds where other actin isoforms are knocked out, thereby enriching for ACT7 as the predominant actin form.

• Immunoprecipitation followed by isoform-specific detection: Use pan-actin antibodies for initial IP, followed by mass spectrometry or isoform-specific antibodies to determine the relative abundance of ACT7 versus other isoforms.

• Super-resolution microscopy: When combined with appropriate controls, advanced imaging techniques may reveal subtle differences in localization patterns between ACT7 and other actin isoforms.

A matrix experimental design comparing results across multiple detection methods and experimental conditions provides the most robust approach to ACT7-specific analysis.

What are common challenges in Western blotting with At1g71900 antibodies and how can they be addressed?

Western blotting with At1g71900 antibodies presents several technical challenges that require specific troubleshooting approaches:

• Multiple banding patterns: Researchers often observe multiple bands due to:

  • Cross-reactivity with other actin isoforms

  • Post-translational modifications of ACT7

  • Degradation products

  Solution: Use freshly prepared samples with protease inhibitors, optimize antibody dilution, and include appropriate controls such as act7 mutants to identify specific bands.

• High background: Non-specific binding can obscure specific signals.

  Solution: Increase blocking stringency (5% BSA instead of milk for phospho-specific detection), optimize antibody concentration, extend washing steps, and consider alternative blocking agents like fish gelatin.

• Weak signal intensity: ACT7 detection may be challenging in tissues with low expression.

  Solution: Increase protein loading, optimize exposure times, use signal enhancement systems (e.g., enhanced chemiluminescence), and consider immunoprecipitation to concentrate the target protein before detection.

• Inconsistent results across experiments: Variation between blots affects reproducibility.

  Solution: Standardize all protocol steps, use internal loading controls, prepare larger batches of working solutions, and develop quantitative normalization procedures.

• Antibody specificity concerns: Distinguishing ACT7 from other actins.

  Solution: Compare results using multiple ACT7 antibody clones (29G12.G5.G6, 33E8.C11.F5.D1, 36H8.C12.H10.B6) , perform peptide competition assays, and validate with genetic approaches.

A systematic approach to optimization, recording all experimental parameters and outcomes, will help establish a reliable protocol for consistent ACT7 detection by Western blotting.

How can researchers optimize immunoprecipitation protocols for At1g71900 antibody-based studies?

Immunoprecipitation (IP) with At1g71900 antibodies can provide valuable insights into ACT7 interactions and modifications. The following methodological considerations will enhance IP success:

• Antibody selection and immobilization:

  • Choose high-affinity At1g71900 antibody clones suitable for IP

  • Consider using Protein G for antibody immobilization, as this purification method has been validated for these antibodies

  • Test both direct antibody immobilization and pre-binding approaches

• Sample preparation:

  • Use gentle lysis buffers (e.g., RIPA or NP-40-based) to preserve protein-protein interactions

  • Include protease and phosphatase inhibitors to prevent degradation and preserve post-translational modifications

  • Optimize extraction conditions based on subcellular localization (cytosolic vs. cytoskeletal-associated fractions)

• Pre-clearing strategy:

  • Pre-clear lysates with beads alone to reduce non-specific binding

  • Consider using species-matched control IgG pre-clearing steps

• Washing optimization:

  • Develop a washing stringency gradient to determine optimal conditions

  • Test different detergent concentrations and salt concentrations to maximize signal-to-noise ratio

• Elution methods:

  • Compare different elution strategies (low pH, high pH, competing peptide, boiling in SDS buffer)

  • For downstream mass spectrometry, consider non-denaturing elution methods

• Validation approaches:

  • Perform reverse IPs with identified interacting partners

  • Include appropriate negative controls (non-specific IgG, lysates from act7 mutants)

  • Confirm specificity with Western blotting of IP products

• Co-IP considerations:

  • For studying ACT7 interactions in auxin signaling contexts, compare IPs from auxin-treated vs. untreated samples

  • Consider crosslinking approaches for transient interactions

Successful IP protocols should be thoroughly documented with all experimental conditions to ensure reproducibility across different biological questions involving ACT7.

What quality control measures should be implemented when working with At1g71900 antibodies?

Implementing rigorous quality control measures ensures reliable and reproducible results when working with At1g71900 antibodies. A comprehensive quality control strategy should include:

• Initial antibody validation:

  • Confirm reactivity against recombinant ACT7 protein

  • Test specificity across multiple applications (WB, ELISA, IF) as relevant to experimental goals

  • Document lot-to-lot variation when receiving new antibody batches

• Regular performance checks:

  • Include consistent positive controls in each experiment

  • Maintain reference samples with known ACT7 expression levels

  • Record and monitor signal-to-noise ratios across experiments

• Antibody storage and handling:

  • Implement proper storage protocols (-20°C in PBS with 0.05% sodium azide)

  • Maintain aliquoting records to track freeze-thaw cycles

  • Regularly test aliquots that have been stored for extended periods

• Application-specific controls:

  • For Western blotting: Include molecular weight markers, loading controls, and tissue-specific expression controls

  • For immunofluorescence: Include secondary-only controls, peptide competition controls, and known expression pattern references

  • For immunoprecipitation: Include IgG controls, input controls, and unbound fraction analyses

• Cross-validation strategies:

  • Compare results across different At1g71900 antibody clones (29G12.G5.G6, 33E8.C11.F5.D1, 36H8.C12.H10.B6)

  • Correlate antibody-based detection with alternative methods (e.g., fluorescent protein fusions, mRNA expression)

  • Document concordance or discordance between different detection methods

• Dilution series optimization:

  • Perform antibody titration experiments for each application

  • Determine optimal signal-to-background ratios

  • Document working concentration ranges for different applications

• Record-keeping systems:

  • Maintain detailed logs of antibody performance

  • Document batch information, storage conditions, and usage history

  • Implement standardized reporting formats for experimental conditions

Establishing these quality control measures as standard laboratory practice will significantly enhance the reliability of At1g71900 antibody-based research and facilitate troubleshooting when issues arise.

How can emerging antibody technologies enhance At1g71900-based research?

Recent advances in antibody technology present new opportunities for studying At1g71900 (ACT7) with increased precision and versatility:

• Single-domain antibodies and nanobodies:

  • Smaller size enables better penetration in plant tissues

  • Potential for direct fusion to fluorescent proteins for live-cell imaging

  • Possibility of developing ACT7 isoform-specific nanobodies with greater epitope discrimination

• Deep learning-based antibody design:

  • Computational approaches similar to those described for therapeutic antibodies could be adapted for generating highly specific plant research antibodies

  • In silico screening could identify optimal epitopes unique to ACT7 among actin isoforms

  • Machine learning algorithms might predict antibody performance characteristics before synthesis

• Recombinant antibody fragments:

  • Fab, scFv, or other engineered formats might provide better access to epitopes in fixed plant tissues

  • Potential for bacterial expression systems to produce renewable, standardized antibody reagents

  • Opportunity for structure-guided engineering to enhance specificity

• Multiplexed detection systems:

  • Development of antibody panels for simultaneous detection of multiple actin isoforms

  • Oligonucleotide-conjugated antibodies for digital counting applications

  • Mass cytometry or imaging mass cytometry approaches for highly multiplexed protein detection

• Proximity labeling applications:

  • Antibody-enzyme fusions for proximity-dependent labeling of ACT7 interaction partners

  • TurboID or APEX2 fusions for mapping the ACT7 interaction network in specific cellular contexts

  • Spatially resolved interactome analysis in different developmental stages

• Intrabodies for in vivo applications:

  • Development of antibodies that function in the reducing environment of plant cytoplasm

  • Potential for targeted manipulation of ACT7 function in specific subcellular domains

Research using these advanced antibody technologies could significantly enhance our understanding of ACT7's role in plant development, auxin responses, and cytoskeletal dynamics.

What are emerging applications for At1g71900 antibodies in plant stress response research?

The relationship between the actin cytoskeleton and plant stress responses represents an expanding research frontier where At1g71900 antibodies could play a crucial role:

• Abiotic stress response mechanisms:

  • Investigating ACT7 remodeling during drought, salinity, temperature, or heavy metal stress

  • Correlating cytoskeletal changes with stress signaling pathways

  • Examining tissue-specific roles of ACT7 in stress adaptation

• Pathogen response dynamics:

  • Tracking ACT7 reorganization during pathogen attack and immune responses

  • Investigating the role of ACT7 in pathogen-triggered endocytosis

  • Exploring connections between hormone signaling, immunity, and ACT7 function

• Cytoskeletal-endomembrane system interactions:

  • Using At1g71900 antibodies to study how ACT7 mediates stress-induced changes in vesicle trafficking

  • Investigating the relationship between ACT7 and autophagy during stress responses

  • Examining organelle movement and positioning governed by ACT7 under stress conditions

• Climate change adaptation research:

  • Studying ACT7 involvement in responses to elevated CO₂, extreme weather events, or UV radiation

  • Investigating potential breeding targets for enhanced stress resilience based on ACT7 function

  • Developing high-throughput screening methods using At1g71900 antibodies to identify stress-resistant variants

• Developmental plasticity under stress:

  • Exploring how ACT7-mediated processes contribute to stress-induced developmental reprogramming

  • Investigating the interface between stress perception and auxin-mediated growth adjustments

  • Studying root architecture modifications mediated by ACT7 under nutrient limitation

• Methodological innovations:

  • Developing ACT7 biosensors based on antibody recognition domains

  • Creating microfluidic systems for real-time monitoring of cytoskeletal responses to controlled stress application

  • Implementing tissue clearing techniques compatible with At1g71900 antibody-based detection for whole-organ imaging

These emerging applications highlight the potential of At1g71900 antibodies as powerful tools for understanding fundamental aspects of plant environmental adaptation mechanisms.