At2g41360 Antibody

What is the At2g41360 gene and why are antibodies against it important?

The At2g41360 gene encodes Actin-7, a critical cytoskeletal protein in Arabidopsis thaliana. Actin-7 has been identified across animals, plants, and protists, with particularly important functions in plant biology. The protein plays essential roles in germination, root growth, and callus tissue formation, making it a significant target for developmental biology research .

Antibodies against At2g41360/Actin-7 provide valuable tools for investigating cytoskeletal dynamics during plant development. They enable researchers to track Actin-7 expression, localization, and interactions in response to various stimuli, particularly plant hormones. The specific monoclonal antibodies developed against A. thaliana Actin-7 offer precise detection in experimental contexts, facilitating research on fundamental plant cellular processes .

What are the primary applications of At2g41360 antibodies in plant research?

At2g41360 antibodies serve multiple critical functions in plant research investigations. These antibodies are validated for Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence (IF) applications, providing diverse experimental options .

In developmental biology, these antibodies allow visualization of actin cytoskeleton remodeling during cell division, expansion, and differentiation. For hormone response studies, they enable tracking of Actin-7's rapid induction following auxin treatment, helping elucidate mechanisms of hormone-induced morphological changes. Additionally, in comparative studies across plant tissues, they facilitate examination of differential expression patterns between rapidly developing versus mature tissues, offering insights into tissue-specific functions of the actin cytoskeleton .

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

Actin-7 distinguishes itself from other plant actin isoforms through several key characteristics. Unlike many constitutively expressed actins, the ACT7 gene demonstrates enhanced expression in rapidly developing tissues and exhibits dynamic responsiveness to external stimuli, particularly phytohormones like auxin .

Functionally, Actin-7 plays specialized roles in germination and root development processes that are not fully compensated by other actin isoforms. Additionally, it is essential for callus tissue formation, suggesting unique functions in dedifferentiation and cellular reprogramming. These distinctive properties make Actin-7 particularly valuable for studying dynamic cytoskeletal responses during plant development and adaptation to environmental conditions .

How can At2g41360 antibodies be used to investigate actin-hormone interactions in signaling pathways?

Investigating actin-hormone interactions requires sophisticated experimental approaches enabled by At2g41360 antibodies. Researchers can employ these antibodies in time-course immunofluorescence studies to visualize dynamic cytoskeletal rearrangements following hormone treatments, particularly tracking the rapid response to auxin exposure . This approach can be complemented with quantitative Western blotting to measure corresponding changes in Actin-7 protein levels.

For more advanced signaling pathway investigations, co-immunoprecipitation experiments using At2g41360 antibodies can identify hormone-dependent interaction partners of Actin-7. Additionally, researchers might combine antibody-based detection with live cell imaging techniques to capture real-time cytoskeletal dynamics. Such multi-technique approaches provide comprehensive insights into how the actin cytoskeleton participates in and responds to hormone signaling cascades that ultimately drive changes in cell morphology and plant development .

What considerations are important when using these antibodies in multiplex immunostaining with other cytoskeletal markers?

Successful multiplex immunostaining with At2g41360 antibodies requires careful experimental planning. When combining these antibodies with markers for other cytoskeletal components (such as microtubules, intermediate filaments, or actin-binding proteins), researchers must first verify the compatibility of fixation and permeabilization protocols for all targets simultaneously .

Cross-reactivity testing is essential to ensure that secondary antibodies do not produce false positive signals. Based on experimental protocols from similar studies, optimal antibody dilutions should be determined separately for single staining before combining in multiplex approaches . Spectral overlap must be carefully addressed when selecting fluorophores for simultaneous visualization; proper compensation settings and sequential scanning approaches may be necessary. For quantitative analyses, researchers should implement standardized image acquisition parameters and appropriate controls for accurate signal normalization across experimental conditions .

How do different epitopes of the At2g41360 protein affect antibody selection for specific experimental conditions?

The epitope recognition properties of different At2g41360 antibody clones (29G12.G5.G6, 33E8.C11.F5.D1, and 36H8.C12.H10.B6) significantly impact experimental outcomes . Different epitopes may become accessible or obscured depending on fixation methods, protein conformation states, or interaction with binding partners.

For optimal experimental design, researchers should consider that conformational epitopes are often better preserved in native conditions for immunoprecipitation studies, while linear epitopes may be more suitable for denatured Western blotting applications. Published research suggests using all three monoclonal antibodies in preliminary experiments to determine which clone performs optimally in specific experimental conditions . This comparative approach is particularly crucial when investigating protein-protein interactions or post-translational modifications that might mask specific epitopes. The accessibility of different epitopes can provide additional structural information about how Actin-7 functions in different cellular compartments or developmental stages .

What are the optimal fixation and sample preparation protocols for At2g41360 antibody applications in different plant tissues?

Different plant tissues require tailored fixation protocols for optimal At2g41360 antibody performance. For immunofluorescence in young, rapidly dividing tissues (where ACT7 is highly expressed), a brief 10-15 minute fixation with 4% paraformaldehyde in PBS effectively preserves cytoskeletal structures while maintaining epitope accessibility . More fibrous or mature tissues might benefit from the addition of 0.1-0.5% glutaraldehyde to enhance structural preservation, though this may require subsequent quenching of autofluorescence with sodium borohydride.

For sectioned material, paraffin embedding should be avoided as it typically destroys actin epitopes; instead, researchers should utilize cryo-sectioning or vibratome sectioning of lightly fixed tissues. When preparing samples for Western blotting, protein extraction buffers should include cytoskeleton-stabilizing components such as phalloidin derivatives to prevent actin depolymerization during extraction. For all applications, researchers should empirically test different fixation times and concentrations with each tissue type, as overfixation frequently leads to epitope masking and reduced antibody binding efficiency .

How should quantitative analysis of At2g41360 expression be performed across different experimental conditions?

Quantitative analysis of At2g41360 expression requires methodological rigor to ensure accurate results across different experimental conditions. For Western blot quantification, researchers should implement loading controls appropriate for plant tissues, such as UBQ10 or TUB4, rather than using conventional housekeeping genes that may fluctuate under experimental treatments . Densitometric analysis should employ linear range determinations for each experiment to ensure measurements fall within the quantifiable range.

For immunofluorescence quantification, standardized image acquisition parameters must be maintained consistently across all samples. Z-stack imaging with defined step sizes followed by maximum intensity projections provides more comprehensive data for cytoskeletal structures. Digital image analysis should employ automated thresholding algorithms to reduce subjective bias, with statistical validation using sufficient biological and technical replicates. For flow cytometry applications, researchers should establish clear gating strategies based on controls and apply consistent compensation between signal channels to account for spectral overlap . All quantitative assessments should include appropriate statistical analyses to determine significance of observed differences.

What approaches can be used to validate At2g41360 antibody specificity in new experimental systems?

Validating At2g41360 antibody specificity in new experimental systems requires multiple complementary approaches. The gold standard validation method is performing parallel experiments with knockout/knockdown mutants (act7 mutants in Arabidopsis) to confirm absence or reduction of signal. Researchers should also conduct pre-adsorption tests by incubating the antibody with purified Actin-7 protein prior to immunostaining to demonstrate specific blocking of the signal .

Western blot analysis should confirm a single band of appropriate molecular weight (~42 kDa for Actin-7) without non-specific binding. When extending application to new plant species, researchers should first perform sequence alignment analysis to confirm sufficient conservation of the epitope region. Cross-reactivity testing against other actin isoforms with known differential expression patterns provides additional specificity confirmation. For systems where genetic manipulation is challenging, an alternative validation approach includes correlation with mRNA expression data (RT-qPCR or in situ hybridization) to verify that protein detection patterns align with transcript distribution .

How can researchers optimize signal-to-noise ratio when using At2g41360 antibodies for immunofluorescence in plant tissues?

Optimizing signal-to-noise ratio for At2g41360 immunofluorescence requires attention to several technical factors. Plant tissues often present unique challenges due to autofluorescence from chlorophyll, cell wall components, and secondary metabolites. Researchers should implement specific blocking steps using a combination of normal serum (5-10%) from the secondary antibody host species and 1-3% BSA in PBS to reduce non-specific binding .

Background reduction can be achieved through careful washing steps (at least 3-4 washes of 10-15 minutes each) with PBS containing 0.1-0.3% Triton X-100. For tissues with high autofluorescence, pre-treatment with 0.1% sodium borohydride or 10mM sodium azide can significantly reduce background. Researchers should also optimize antibody concentrations through titration experiments, typically testing dilutions ranging from 1:100 to 1:5000, to identify the concentration that provides maximum specific signal with minimal background. When possible, confocal microscopy with appropriate filter settings helps distinguish specific signal from autofluorescence through spectral separation .

What are common pitfalls when interpreting Western blot results using At2g41360 antibodies?

Interpreting Western blot results with At2g41360 antibodies presents several potential pitfalls that researchers should carefully address. A common misinterpretation occurs when multiple bands appear - researchers should recognize that plant actins share high sequence homology (>90% in some regions), potentially leading to cross-reactivity with other actin isoforms . Additionally, post-translational modifications of Actin-7 (such as phosphorylation, ubiquitination, or acetylation) can cause mobility shifts that may be misinterpreted as non-specific binding.

Another frequent issue arises from degradation products appearing as lower molecular weight bands, particularly when sample preparation does not include appropriate protease inhibitors. Researchers should also be cautious about quantitative interpretations when comparing tissues with different extraction efficiencies or when signal intensity exceeds the linear range of detection. It is recommended to include positive controls (tissues known to express high levels of Actin-7) and negative controls (actin-7 knockout tissue when available) in experimental designs . Finally, proper loading controls that remain stable under experimental conditions are essential for accurate quantitative analyses.

How can researchers determine the appropriate antibody concentration for different experimental applications?

Determining optimal antibody concentration requires systematic titration across different applications. For Western blotting applications, researchers should prepare a dilution series typically ranging from 1:500 to 1:5000 of the primary antibody against a constant amount of protein extract containing Actin-7. The ideal concentration provides clear specific bands with minimal background at the shortest exposure time .

For immunofluorescence applications, the recommended approach is preparing serial dilutions between 1:100 and 1:1000 while maintaining consistent secondary antibody concentration, fixation, and imaging parameters. The optimal concentration should provide sufficient signal intensity while minimizing background fluorescence. For ELISA applications, a checkerboard titration approach is recommended, where both primary antibody and antigen concentrations are varied systematically to identify optimal signal-to-noise ratios .

Different applications typically require different antibody concentrations - ELISA often requires higher concentrations than Western blotting, while immunoprecipitation may require significantly more antibody than immunofluorescence. Researchers should note that the three available monoclonal antibody clones (29G12.G5.G6, 33E8.C11.F5.D1, 36H8.C12.H10.B6) may have different optimal working concentrations, with published protocols recommending initial testing of all three to determine which performs best for each specific experimental setup .

How can At2g41360 antibodies be integrated with proteomics approaches to study actin-interacting proteins?

Integrating At2g41360 antibodies with proteomics offers powerful approaches for investigating actin interaction networks. Researchers can employ co-immunoprecipitation (Co-IP) using At2g41360 antibodies coupled to protein A/G beads to pull down Actin-7 along with its binding partners from plant extracts . This approach can be enhanced through crosslinking protocols that stabilize transient interactions prior to extraction. The precipitated protein complexes can then be analyzed using mass spectrometry to identify novel interaction partners.

Proximity-dependent biotin labeling techniques represent another advanced approach, where At2g41360 antibodies can be used to validate the localization of identified interaction partners through co-localization studies. For temporal analyses of dynamic interaction networks, researchers can combine At2g41360 antibody-based pulldowns with SILAC (Stable Isotope Labeling with Amino acids in Cell culture) or TMT (Tandem Mass Tag) labeling to quantify changes in protein-protein interactions across developmental stages or in response to hormonal treatments . These integrated approaches provide comprehensive insights into the functional roles of Actin-7 within broader cytoskeletal regulatory networks.

What considerations are important when designing FRET/FLIM experiments involving labeled At2g41360 antibodies?

Designing FRET/FLIM (Förster Resonance Energy Transfer/Fluorescence Lifetime Imaging Microscopy) experiments with labeled At2g41360 antibodies requires careful planning to obtain reliable protein interaction data. Researchers must first confirm that fluorophore conjugation to the antibodies does not impair antigen recognition, which can be verified through parallel immunofluorescence experiments with labeled and unlabeled antibodies .

The choice of fluorophore pairs is critical - typical donor/acceptor pairs such as Alexa Fluor 488/Alexa Fluor 555 or CFP/YFP should have appropriate spectral overlap while minimizing bleed-through. For antibody labeling, researchers should target a fluorophore-to-protein ratio of 2-4 molecules per antibody to avoid self-quenching effects. Controls must include donor-only samples to establish baseline fluorescence lifetime values and negative interaction controls using antibodies against non-interacting proteins .

When interpreting FRET/FLIM data, researchers should consider that the large size of antibodies (~150 kDa) may place fluorophores at distances exceeding the optimal FRET range (typically 1-10 nm), potentially reducing signal detection for true interactions. Alternative approaches like using fluorescently-labeled Fab fragments or nanobodies can sometimes provide improved spatial resolution by reducing the distance between fluorophores in genuine interaction scenarios .

What statistical approaches are most appropriate for analyzing quantitative data from At2g41360 antibody experiments?

Selecting appropriate statistical methods for At2g41360 antibody experiment data requires consideration of experimental design and data distribution. For comparing Actin-7 expression levels across experimental treatments (such as hormone exposure or developmental stages), researchers should first assess data normality using Shapiro-Wilk or Kolmogorov-Smirnov tests. Normally distributed data from independent groups can be analyzed using t-tests (for two groups) or ANOVA followed by post-hoc tests (for multiple groups) .

For non-normally distributed data, non-parametric alternatives such as Mann-Whitney U or Kruskal-Wallis tests are more appropriate. When analyzing time-course experiments of Actin-7 expression, repeated measures ANOVA or mixed-effects models should be employed to account for within-subject correlations. Co-localization analyses between Actin-7 and other proteins require correlation coefficients such as Pearson's or Mander's, depending on whether linear relationships or overlap is being assessed .

Power analysis should be conducted prior to experiments to determine appropriate sample sizes for detecting biologically meaningful differences. For all analyses, researchers should report effect sizes alongside p-values to communicate biological significance in addition to statistical significance. Multiple testing corrections (such as Bonferroni or FDR) must be applied when performing numerous comparisons to control false discovery rates .

How should researchers interpret changes in At2g41360 localization patterns in response to experimental treatments?

Interpreting changes in Actin-7 localization patterns requires systematic approaches to distinguish meaningful biological responses from technical artifacts. Researchers should establish baseline localization patterns in control conditions through Z-stack confocal imaging to capture the three-dimensional distribution of Actin-7 within cells . Quantitative parameters such as filament density, bundling, orientation, and co-localization with cellular markers should be measured using specialized image analysis software.

When evaluating treatment effects, researchers must consider both spatial and temporal dimensions of localization changes. Rapid rearrangements (within minutes) may indicate direct effects on actin dynamics, while slower changes (hours to days) might reflect transcriptional or translational regulation. Particular attention should be paid to redistribution between different cellular compartments, formation of specific structures (such as actin patches or cables), or changes in cytoskeletal-membrane associations .

Biological interpretation should consider known functions of Actin-7 in specific contexts - for example, relocalization to cell expansion zones following auxin treatment would be consistent with Actin-7's role in cell elongation. To establish causality between localization changes and biological outcomes, complementary approaches such as pharmacological disruption of actin dynamics or genetic manipulation should be employed alongside antibody-based visualization .

Comparative Analysis Table of At2g41360 Antibody Clones

Note: Table compiled based on available research data. Optimal conditions may vary based on specific experimental context. Initial testing of all three antibodies is recommended for new experimental systems .