At2g39490 Antibody

What is the At2g39490 protein and why is it significant in plant research?

At2g39490 is an F-box protein in Arabidopsis thaliana (Mouse-ear cress) that functions within the ubiquitin-proteasome system. F-box proteins typically serve as substrate recognition components within SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase complexes, targeting specific proteins for degradation. This protein may be involved in regulatory pathways similar to those of DELLA proteins, which are GA-signaling repressors that influence plant development. Understanding At2g39490 function contributes to our knowledge of protein degradation pathways in plants and potentially how these systems regulate growth and development in response to environmental cues .

What are the optimal storage conditions for At2g39490 antibodies?

At2g39490 antibodies should be stored at -20°C or -80°C immediately upon receipt to maintain reactivity and specificity. Repeated freeze-thaw cycles should be avoided as they can significantly degrade antibody quality. For working solutions, storage at 4°C for up to one month is acceptable, but longer-term storage requires freezing. The antibodies are typically supplied in a liquid format containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . These conditions help maintain antibody stability and prevent microbial contamination during storage periods.

How does At2g39490 relate to plant hormone signaling pathways?

While specific information about At2g39490's role in hormone signaling is limited in the search results, its identity as an F-box protein suggests potential involvement in hormone-mediated developmental regulation. In Arabidopsis, many F-box proteins participate in hormone signaling pathways, including auxin (TIR1/AFBs), jasmonate (COI1), and gibberellin (SLY1/SNE) responses. F-box proteins often target transcriptional regulators for degradation in response to hormone perception. Given the information about DELLA proteins and GA signaling in related research, At2g39490 might function in similar regulatory networks . DELLA proteins are degraded via the 26S proteasome pathway in response to GA, which involves F-box proteins recognizing their targets after hormone-induced conformational changes.

What immunoprecipitation protocols are most effective with At2g39490 antibodies?

For optimal immunoprecipitation using At2g39490 antibodies, researchers should employ protocols similar to those used for other plant protein immunoprecipitations. Based on methodologies described for similar plant protein studies, a recommended approach includes: 1) Homogenizing plant tissue in extraction buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 0.1% NP-40, and protease inhibitor cocktail; 2) Clearing lysates by centrifugation (14,000 rpm, 10 minutes, 4°C); 3) Pre-clearing with Protein A/G beads; 4) Incubating cleared lysates with At2g39490 antibody (2-5 μg) overnight at 4°C; 5) Adding fresh Protein A/G beads for 2-3 hours; 6) Washing beads 4-5 times with wash buffer; and 7) Eluting bound proteins with SDS sample buffer for analysis . For studying potential ubiquitination, including MG132 (100 μM) in the extraction buffer is essential to prevent proteasomal degradation of ubiquitinated substrates.

How can researchers validate the specificity of At2g39490 antibodies?

Validating antibody specificity for At2g39490 requires multiple complementary approaches. First, Western blot analysis should show a single band at approximately 44 kDa in wild-type Arabidopsis samples, which should be absent or significantly reduced in At2g39490 knockout/knockdown lines. Second, immunoprecipitation followed by mass spectrometry can confirm that the antibody pulls down At2g39490 protein. Third, immunohistochemistry results should be compared with known expression patterns of At2g39490 from transcriptomic data. Finally, competition assays using recombinant At2g39490 protein to block antibody binding provide additional validation . For negative controls, researchers should include samples from knockout mutants and use non-immune IgG from the same species as the antibody (rabbit) to establish baseline non-specific binding.

What is the recommended protocol for immunofluorescence detection of At2g39490 in plant tissues?

For immunofluorescence localization of At2g39490 in plant tissues, researchers should: 1) Fix freshly harvested tissue in 4% paraformaldehyde in PBS for 30-60 minutes under vacuum; 2) Wash tissues three times with PBS; 3) Prepare sections (10-20 μm) using a vibratome or microtome; 4) Permeabilize with 0.1-0.5% Triton X-100 in PBS for 15-30 minutes; 5) Block with 2-5% BSA or normal goat serum in PBS for 1 hour; 6) Incubate with At2g39490 primary antibody (diluted 1:100 to 1:500 in blocking solution) overnight at 4°C; 7) Wash three times with PBS; 8) Incubate with fluorophore-conjugated secondary antibody (anti-rabbit) for 1-2 hours at room temperature; 9) Counterstain with DAPI to visualize nuclei; and 10) Mount and image using confocal microscopy . Controls should include omission of primary antibody and comparison with At2g39490 mutant tissues to verify signal specificity.

How can At2g39490 antibodies be used to study protein-protein interactions in ubiquitin-proteasome pathways?

At2g39490 antibodies can be powerful tools for investigating protein-protein interactions within ubiquitin-proteasome pathways through several advanced techniques. Co-immunoprecipitation (Co-IP) using At2g39490 antibodies can identify proteins that interact with this F-box protein within its native SCF complex. This approach should be combined with proteasome inhibitor treatment (e.g., 100 μM MG132) to stabilize transient interactions . For studying dynamic interactions, researchers can perform time-course experiments after treatment with relevant plant hormones or environmental stimuli. Proximity-dependent biotin identification (BioID) or APEX2 proximity labeling coupled with At2g39490 antibody validation can map the protein's interaction neighborhood. Additionally, chromatin immunoprecipitation (ChIP) can be adapted to investigate if At2g39490 associates with specific chromatin regions through interaction with transcription factors, similar to analyses performed for other regulatory pathways in Arabidopsis .

What approaches can be used to investigate the role of At2g39490 in hormone-mediated developmental regulation?

Investigating At2g39490's role in hormone-mediated developmental regulation requires multifaceted approaches combining genetic, biochemical, and immunological techniques. Researchers should create transgenic Arabidopsis lines expressing tagged versions of At2g39490 (TAP-tag, YFP-tag) to track protein behavior in vivo, similar to approaches used for DELLA protein studies . Treating plants with different hormones (gibberellins, auxins, etc.) followed by immunoprecipitation with At2g39490 antibodies can reveal hormone-dependent changes in protein interactions or post-translational modifications. Comparative proteomics between wild-type and At2g39490 mutant plants under different hormone treatments can identify potential substrates. ChIP-seq using At2g39490 antibodies may identify genomic regions associated with At2g39490-containing complexes. Crucially, researchers should correlate At2g39490 protein levels (detected by immunoblotting) with specific developmental phenotypes to establish causative relationships between protein function and plant development.

How can researchers use At2g39490 antibodies to analyze post-translational modifications?

To analyze post-translational modifications (PTMs) of At2g39490, researchers should employ a combination of immunoprecipitation using At2g39490 antibodies followed by specific PTM detection techniques. For ubiquitination analysis, immunoprecipitation of At2g39490 followed by immunoblotting with anti-ubiquitin antibodies can detect ubiquitinated forms, similar to methods used for analyzing DELLA protein ubiquitination . High-molecular-weight protein species detected after immunoprecipitation may indicate multi-ubiquitinated forms. For phosphorylation studies, immunoprecipitated At2g39490 can be analyzed by phospho-specific antibodies or mass spectrometry. Phosphorylation site mutants can be generated and their protein stability assessed using At2g39490 antibodies. Additionally, phos-tag SDS-PAGE followed by immunoblotting with At2g39490 antibodies can separate different phosphorylated forms of the protein to examine how phosphorylation states change under different conditions or treatments.

What are common issues when using At2g39490 antibodies and how can they be resolved?

Researchers commonly encounter several issues when working with At2g39490 antibodies. First, weak or absent signals in Western blots may occur due to low protein expression or antibody degradation. This can be addressed by increasing protein loading (50-100 μg total protein), optimizing extraction buffers with additional protease inhibitors, and ensuring proper antibody storage. Second, high background in immunostaining can be reduced by increasing blocking time (2-3 hours), using 5% BSA instead of normal serum, and extending wash steps . Third, non-specific bands in immunoblots can be minimized by increasing antibody dilution (1:1000 to 1:5000) and optimizing blocking conditions. Fourth, for small volume antibody vials that become entrapped in the seal during shipment, briefly centrifuging the vial before opening can help recover the material . Finally, inconsistent immunoprecipitation results can be improved by pre-clearing lysates more thoroughly and increasing the amount of antibody used (3-5 μg per reaction).

How should researchers interpret conflicting results between At2g39490 antibody data and genetic analyses?

When faced with discrepancies between antibody-based data and genetic analyses of At2g39490, researchers should systematically evaluate several potential sources of conflict. First, examine antibody specificity through additional validation experiments, including Western blots comparing wild-type and knockout tissues. Second, consider potential genetic compensation mechanisms in knockout lines that may mask phenotypes; analyze expression of closely related F-box protein genes in At2g39490 mutants. Third, evaluate whether the antibody recognizes specific post-translationally modified forms of At2g39490 that may not correlate directly with transcript levels. Fourth, examine temporal and spatial expression patterns, as whole-tissue analyses may obscure cell-type-specific functions. Fifth, consider protein stability factors; At2g39490 may have a different half-life than its transcript. Finally, perform complementation experiments with tagged At2g39490 versions in knockout backgrounds to confirm that antibody-detected protein correlates with functional rescue . These approaches will help determine whether discrepancies represent technical artifacts or biologically relevant phenomena.

What statistical approaches are appropriate for quantifying At2g39490 protein levels across different experimental conditions?

Appropriate statistical analysis of At2g39490 protein levels requires careful experimental design and application of suitable quantitative methods. For Western blot quantification, normalization to multiple housekeeping proteins (such as CAND1, used as a control in similar studies) is essential for reliable comparisons . Researchers should perform at least three biological replicates and use densitometry software to quantify band intensities. For comparative analyses across multiple conditions, ANOVA with post-hoc tests (Tukey's or Dunnett's) is appropriate when assumptions of normality are met; otherwise, non-parametric alternatives like Kruskal-Wallis should be used. For time-course experiments, repeated measures ANOVA or mixed-effects models provide more power. When analyzing co-localization in immunofluorescence studies, Pearson's or Mander's correlation coefficients should be calculated from multiple images across different samples. For all analyses, researchers should report effect sizes alongside p-values to indicate biological significance and use appropriate correction methods (e.g., Benjamini-Hochberg) when making multiple comparisons to control false discovery rates.

How does At2g39490 function compare to similar F-box proteins in other plant species?

The function of At2g39490 should be examined in an evolutionary context by comparing it with homologous F-box proteins across plant species. While specific comparative data is not provided in the search results, researchers can apply approaches similar to those used in studying conserved plant signaling components. F-box proteins often show functional conservation despite sequence divergence, particularly in substrate recognition domains. Researchers should perform phylogenetic analyses to identify true orthologs versus paralogs of At2g39490 in crops and other model plants. Cross-species complementation experiments, where the At2g39490 gene from Arabidopsis is expressed in mutants of other species (and vice versa), can reveal functional conservation. Immunoprecipitation with At2g39490 antibodies followed by mass spectrometry in different species can identify conserved interacting partners . Comparative expression analyses during developmental processes and in response to environmental stresses can further illuminate conserved functions. This evolutionary perspective is crucial for translating fundamental research on At2g39490 to applications in agriculturally important plants.

What are the implications of At2g39490 research for understanding plant responses to environmental stresses?

At2g39490 research has potential implications for understanding plant stress responses, particularly if this F-box protein participates in hormone-regulated pathways similar to the DELLA protein signaling network. DELLA proteins function at the intersection of multiple stress response pathways and growth regulation . If At2g39490 operates in related pathways, it may similarly integrate environmental signals with developmental programs. Researchers should examine At2g39490 protein levels (using the specific antibody) under various stress conditions such as drought, salinity, temperature extremes, and pathogen infection. Changes in At2g39490 localization, detected through immunofluorescence, may indicate stress-specific functions. Comparative proteomics between wild-type and At2g39490 mutant plants under stress conditions can reveal downstream targets relevant to stress adaptation. Understanding how At2g39490 participates in stress responses could potentially inform breeding or engineering strategies for improved crop resilience, particularly if conserved mechanisms exist in agricultural species.

How might research on At2g39490 contribute to broader understanding of regulatory networks in plant development?

Research on At2g39490 has the potential to expand our understanding of regulatory networks governing plant development, particularly if it intersects with established hormone signaling pathways. F-box proteins like At2g39490 often function as critical nodes in regulatory networks by controlling the stability of key transcription factors or signaling components. Using At2g39490 antibodies in combination with chromatin immunoprecipitation and protein interaction studies can help map its position within developmental regulatory networks . As demonstrated in research on DELLA proteins, which regulate Arabidopsis development through interaction with various transcription factors and chromatin remodeling complexes, similar approaches can be applied to At2g39490 . Systems biology approaches that integrate At2g39490 protein interaction data, transcript profiles from mutants, and developmental phenotypes can position this protein within the broader signaling landscape. Such integrated understanding contributes to fundamental knowledge of plant developmental biology and potentially identifies new targets for crop improvement strategies.