At4g33900 Antibody

What is At4g33900 protein and what is its function in Arabidopsis thaliana?

At4g33900 is an F-box/kelch-repeat protein found in Arabidopsis thaliana, identified by the UniProt accession number Q1PE27. F-box proteins typically function as components of SCF (Skp1, Cullin, F-box) E3 ubiquitin ligase complexes, which are responsible for targeting specific proteins for ubiquitination and subsequent degradation by the 26S proteasome. The kelch-repeat domain likely mediates protein-protein interactions, potentially determining substrate specificity. In Arabidopsis, F-box proteins like At4g33900 often regulate important developmental processes, stress responses, and hormone signaling pathways through controlled protein turnover. The specific substrates and physiological roles of At4g33900 continue to be active areas of investigation in plant molecular biology.

What forms of At4g33900 antibody are available for research applications?

At4g33900 antibodies are typically available in polyclonal form, as indicated by commercial suppliers like THE BioTek (catalog: BT2471258). These antibodies are generated by immunizing host animals with specific At4g33900 protein fragments or synthetic peptides derived from the protein sequence. While the search results do not specifically mention monoclonal antibodies targeting At4g33900, the field of plant antibodies has been expanding to include diverse formats. Given recent advances in antibody technology, researchers might also consider exploring the development of nanobodies (single-domain antibodies) against At4g33900, similar to approaches used for other proteins . The choice between polyclonal, monoclonal, or alternative antibody formats would depend on the specific research application and required specificity.

What are the recommended storage conditions for At4g33900 antibody?

At4g33900 antibodies are typically supplied in liquid form with specific buffer compositions designed to maintain stability and activity. The antibody from THE BioTek is preserved in a solution containing 0.03% ProClin 300 as a preservative, 50% Glycerol, and 0.01M Phosphate-Buffered Saline (PBS) at pH 7.4. For shipping, these antibodies are transported with ice packs to maintain low temperature. While specific storage temperature information isn't provided in the search results, typical antibody storage recommendations include keeping aliquoted samples at -20°C for long-term storage and avoiding repeated freeze-thaw cycles, which can degrade antibody performance. For working solutions, storage at 4°C for limited periods (typically 1-2 weeks) is generally acceptable, but researchers should consult product-specific documentation for optimal conditions.

How can At4g33900 antibody be used to study F-box protein-mediated protein degradation pathways?

At4g33900 antibody serves as a valuable tool for investigating F-box protein-mediated degradation pathways through multiple experimental approaches. Co-immunoprecipitation (Co-IP) using At4g33900 antibody can identify interaction partners within the SCF complex (including Skp1 and Cullin homologs) and potential substrate proteins targeted for degradation. When combined with mass spectrometry, this approach can reveal the protein interaction network surrounding At4g33900. Additionally, researchers can employ the antibody in ubiquitination assays to detect ubiquitinated substrates of At4g33900 in vitro or in vivo. For temporal regulation studies, the antibody can track At4g33900 protein levels during different developmental stages or stress responses using Western blotting. Immunohistochemistry with the antibody can reveal spatial localization patterns within plant tissues, providing insights into where these degradation pathways operate. When used in chromatin immunoprecipitation (ChIP) experiments, the antibody might help determine if At4g33900 associates with chromatin or transcriptional machinery, expanding our understanding of F-box protein functions beyond protein degradation.

What methodological considerations are important when using antibodies for studying Arabidopsis proteins?

When working with antibodies targeting Arabidopsis proteins like At4g33900, researchers must consider several methodological factors to ensure reliable results. Arabidopsis, as a model organism in plant biology, presents specific challenges and opportunities for antibody-based research . First, antibody specificity must be rigorously validated, ideally using knockout mutants of At4g33900 as negative controls to confirm absence of signal. Second, sample preparation techniques are crucial—plant tissues contain various compounds that can interfere with antibody binding, necessitating optimized extraction buffers containing appropriate detergents, reducing agents, and protease inhibitors. Third, researchers should consider tissue-specific expression patterns of At4g33900, as protein abundance may vary significantly across different plant tissues and developmental stages, requiring careful selection of experimental material. Fourth, subcellular fractionation may be necessary to enrich for the compartment where At4g33900 is predominantly located. Finally, appropriate positive controls should be included, such as recombinant At4g33900 protein of known concentration, to establish detection limits and quantification parameters.

What are the optimal conditions for using At4g33900 antibody in Western blotting?

For optimal Western blotting results with At4g33900 antibody, researchers should consider the following protocol parameters. Sample preparation should include an extraction buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and protease inhibitor cocktail to preserve protein integrity. For protein separation, an 8-12% SDS-PAGE gel is typically suitable for F-box proteins like At4g33900, which is approximately 40-45 kDa. Transfer to nitrocellulose or PVDF membranes should be performed at 100V for 1 hour or 30V overnight at 4°C. For blocking, 5% non-fat dry milk or 3% BSA in TBST (TBS with 0.1% Tween-20) for 1 hour at room temperature is recommended to minimize background. The primary antibody incubation should be performed at dilutions between 1:500 to 1:2000 (optimized by titration) in blocking buffer overnight at 4°C. After washing with TBST (3 × 10 minutes), an appropriate secondary antibody conjugated to HRP should be applied at 1:5000 to 1:10000 dilution for 1 hour at room temperature. Following final washes, detection can be performed using enhanced chemiluminescence (ECL) reagents. Inclusion of positive controls (recombinant At4g33900) and negative controls (At4g33900 knockout plant extracts) is essential for result interpretation.

How can researchers validate At4g33900 antibody specificity for their experimental system?

Validating antibody specificity is critical for reliable research with At4g33900 antibody. A comprehensive validation approach includes multiple complementary methods. First, researchers should perform Western blotting using wild-type Arabidopsis extracts alongside extracts from At4g33900 knockout or knockdown lines—a specific antibody will show significantly reduced or absent signal in the mutant samples. Second, preabsorption tests can be conducted by pre-incubating the antibody with excess purified recombinant At4g33900 protein before immunostaining or Western blotting; specific antibodies will show diminished signal after preabsorption. Third, ectopic expression systems can be utilized by overexpressing tagged versions of At4g33900 in Arabidopsis or heterologous systems, then confirming co-detection with both tag-specific antibodies and the At4g33900 antibody. Fourth, mass spectrometry analysis of immunoprecipitated proteins can verify that the antibody captures the intended target. Finally, cross-reactivity testing against closely related F-box proteins can assess potential off-target binding. Researchers should document these validation steps thoroughly and consider that antibody performance may vary across different experimental techniques (Western blotting, immunoprecipitation, immunofluorescence).

How should researchers address non-specific binding issues with At4g33900 antibody?

Non-specific binding is a common challenge when working with antibodies, including those targeting At4g33900. To address this issue, researchers can implement several optimization strategies. First, increase the stringency of blocking by using 5% BSA instead of milk proteins, or adding 0.1-0.3% Tween-20 to reduce hydrophobic interactions. Second, optimize antibody dilution through careful titration experiments to find the concentration that maximizes specific signal while minimizing background. Third, increase the number and duration of washing steps (4-6 washes of 10-15 minutes each) with TBST containing up to 0.3% Tween-20 for stubborn background issues. Fourth, consider alternative blocking agents such as commercial blocking reagents specifically designed for plant samples, which may better prevent non-specific interactions in Arabidopsis extracts. Fifth, pre-adsorb the antibody with extracts from At4g33900 knockout plants to remove antibodies that bind to non-target proteins. Sixth, for immunohistochemistry applications, include an additional blocking step with endogenous peroxidase quenching (3% hydrogen peroxide in methanol) and avidin/biotin blocking if biotin-based detection systems are used. If these adjustments don't resolve non-specific binding, consider testing alternative lots or sources of At4g33900 antibody, as antibody quality can vary between manufacturing batches.

How can researchers interpret contradictory results from different detection methods using At4g33900 antibody?

When faced with contradictory results from different detection methods using At4g33900 antibody, researchers should systematically evaluate several factors. First, consider epitope accessibility—the protein conformation may differ between methods (denatured in Western blotting versus native in immunoprecipitation), affecting antibody binding. Second, evaluate method sensitivity thresholds, as techniques like Western blotting with chemiluminescence detection might detect lower protein amounts than immunofluorescence. Third, examine fixation effects, as certain fixatives used in immunohistochemistry might mask or alter the antibody epitope. Fourth, assess buffer compatibility, since extraction and assay buffers vary between techniques and might affect antibody-antigen interactions. Fifth, investigate post-translational modifications that might be differentially present in samples prepared for different techniques, potentially affecting antibody recognition. Sixth, compare subcellular localization results carefully, as fractionation methods might enrich or deplete certain compartments, leading to apparent contradictions. To resolve discrepancies, researchers should implement cross-validation approaches using complementary techniques (e.g., mass spectrometry of immunoprecipitated samples), consider using multiple antibodies targeting different epitopes of At4g33900, and evaluate if results align with known biology of similar F-box proteins. Documenting all experimental conditions meticulously will help identify variables contributing to contradictory outcomes.

What potential exists for developing nanobodies against At4g33900 for in vivo studies?

The development of nanobodies against At4g33900 presents an exciting frontier for advancing in vivo studies of this F-box protein. Nanobodies, derived from camelid heavy-chain-only antibodies, offer several advantages over conventional antibodies that make them particularly promising for plant research . Their small size (approximately 15 kDa, compared to 150 kDa for conventional antibodies) enables superior tissue penetration and access to epitopes that might be inaccessible to larger antibodies . Nanobodies' exceptional stability allows them to function in the reducing environment of plant cells, maintaining activity where conventional antibodies might denature . For At4g33900 research, nanobodies could enable real-time tracking of protein dynamics by fusing them with fluorescent proteins to create "chromobodies" that bind the native protein without requiring genetic modification of the target. The relatively simple genetic structure of nanobodies also facilitates their expression directly within plant cells as "intrabodies," potentially enabling visualization or even manipulation of At4g33900 function in specific tissues or developmental stages. Following the successful approach used at the University of Kentucky for developing nanobodies against cancer-related proteins, researchers could immunize alpacas with purified At4g33900 protein, collect blood samples after six weeks, and isolate the nanobodies targeting the protein .

How might comparative studies of At4g33900 orthologs in other plant species advance our understanding?

Comparative studies of At4g33900 orthologs across diverse plant species represent a powerful approach to elucidating this protein's evolutionary conservation, functional significance, and species-specific adaptations. By employing At4g33900 antibody alongside antibodies developed against orthologous proteins from other species, researchers can investigate whether functional domains and interaction partners are conserved across evolutionary distances. Such cross-species immunoprecipitation studies might reveal conserved core complexes versus species-specific interactions that have evolved to address particular environmental challenges. Additionally, examining expression patterns of At4g33900 orthologs in crop plants versus Arabidopsis could provide insights into how this F-box protein's function may have been modified during domestication or adaptation to different ecological niches . For species where genetic manipulation is challenging, antibody-based approaches may be particularly valuable for studying protein function. To maximize cross-reactivity potential, researchers should focus antibody development on highly conserved regions of the protein identified through bioinformatic analysis. Complementing antibody-based approaches with comparative genomics and transcriptomics will provide a multi-dimensional understanding of how At4g33900-like proteins function across the plant kingdom, potentially revealing novel applications for improving crop stress resistance or yield.

What is the recommended protocol for immunoprecipitation with At4g33900 antibody?

For successful immunoprecipitation (IP) of At4g33900 and its interaction partners, researchers should follow this optimized protocol. Begin with fresh plant tissue (preferably 1-2 g) and grind in liquid nitrogen to a fine powder. Extract proteins in ice-cold IP buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100 or 0.5% NP-40, 1 mM EDTA, 10% glycerol) supplemented with protease inhibitor cocktail, 1 mM DTT, and phosphatase inhibitors if phosphorylation studies are planned. After centrifugation at 14,000g for 15 minutes at 4°C, pre-clear the supernatant with Protein A/G beads for 1 hour at 4°C with gentle rotation. For the IP reaction, incubate 1-5 µg of At4g33900 antibody with pre-cleared lysate overnight at 4°C with gentle rotation. Add 30-50 µl of Protein A/G magnetic beads and incubate for an additional 2-3 hours. Perform washing steps with increasingly stringent buffers: twice with IP buffer, twice with IP buffer containing 300 mM NaCl, and once with 50 mM Tris-HCl pH 7.5. Elute bound proteins by boiling in SDS-PAGE sample buffer for direct analysis, or use a gentler elution with peptide competition for functional studies. Critical controls should include a non-immune IgG antibody of the same species and ideally a parallel IP from At4g33900 knockout plants. For detecting rare or transient interactions, consider crosslinking with 1% formaldehyde prior to extraction, or including proteasome inhibitors (MG132) in the growth medium before harvest.