At3g58950 Antibody

What is the AT3G58950 gene and its encoded protein in Arabidopsis thaliana?

The AT3G58950 gene encodes an F-box/RNI-like superfamily protein in Arabidopsis thaliana (mouse-ear cress) . F-box proteins are characterized by the presence of an F-box motif and typically function as components of SCF (Skp1-Cullin-F-box) ubiquitin ligase complexes, which play crucial roles in protein degradation via the ubiquitin-proteasome pathway. Within the plant cellular context, F-box proteins often regulate various developmental processes and responses to environmental stimuli through targeted protein degradation. The specific molecular mechanisms and biological functions of the AT3G58950-encoded protein remain under investigation, making antibodies against this protein valuable tools for elucidating its roles within plant cellular processes.

What are the key characteristics of the At3g58950 Antibody?

The At3g58950 Antibody (e.g., product code CSB-PA878629XA01DOA) is a polyclonal antibody raised in rabbits against a recombinant Arabidopsis thaliana At3g58950 protein . It is supplied in liquid form with 50% glycerol in a 0.01M PBS (pH 7.4) buffer containing 0.03% Proclin 300 as a preservative . The antibody undergoes antigen affinity purification to enhance specificity . It has been tested for applications including ELISA and Western Blotting (WB), making it suitable for protein detection and quantification experiments . As a research-grade reagent, it is designated for research use only and not intended for diagnostic or therapeutic procedures . The polyclonal nature of this antibody means it recognizes multiple epitopes on the target protein, potentially providing robust detection but requiring careful validation to ensure specificity.

How should At3g58950 Antibody be stored and handled for optimal performance?

Upon receipt, the At3g58950 Antibody should be stored at -20°C or -80°C for long-term stability . Researchers should avoid repeated freeze-thaw cycles as these can degrade antibody quality and compromise experimental results. For working solutions, antibody aliquots can be prepared in small volumes to minimize freeze-thaw cycles. Before each use, the antibody should be gently mixed by inversion rather than vortexing to avoid protein denaturation. When preparing dilutions, use high-quality, sterile buffers appropriate for the intended application. Documentation of storage conditions, freeze-thaw cycles, and dilution factors is essential for experimental reproducibility. Proper handling includes wearing appropriate personal protective equipment and maintaining aseptic technique to prevent microbial contamination that could affect antibody performance or introduce experimental artifacts.

What is the expected lead time for obtaining At3g58950 Antibody?

The At3g58950 Antibody is typically made-to-order with a lead time of approximately 14-16 weeks . This extended production period reflects the complex processes involved in generating and validating research-grade antibodies. Researchers should plan experiments accordingly, incorporating this substantial lead time into project timelines. For time-sensitive projects, it is advisable to order well in advance of anticipated experimental start dates. Additionally, researchers might consider exploring whether small aliquots of pre-validated antibody are available for preliminary studies while awaiting delivery of a complete order. The lengthy production process underscores the importance of proper antibody storage and handling upon receipt to maximize utility and prevent waste of this valuable, time-intensive reagent.

How should At3g58950 Antibody be validated before experimental use?

Before employing At3g58950 Antibody in critical experiments, comprehensive validation is essential to ensure specificity and reliability. Begin with Western blot analysis using positive control (Arabidopsis thaliana extract) and negative control (unrelated plant species) samples to confirm specificity for the target protein . The antibody should detect a band of the expected molecular weight for the At3g58950 protein. Perform peptide competition assays by pre-incubating the antibody with excess immunizing peptide prior to application; this should significantly reduce or eliminate specific signal if the antibody is target-specific . For immunohistochemistry applications, parallel staining with pre-immune serum can identify potential background or non-specific binding issues. Additionally, validate the antibody in knockout or knockdown plant materials where available, as absence or reduction of signal would further confirm specificity. Document all validation experiments thoroughly, including antibody dilutions, exposure times, and relevant experimental conditions to establish reproducible protocols.

What techniques can At3g58950 Antibody be successfully applied to in plant research?

At3g58950 Antibody has been successfully tested for ELISA and Western Blot applications , but researchers can potentially adapt it for other immunological techniques with proper validation. For Western blotting, the antibody can detect At3g58950 protein in plant tissue extracts following standard SDS-PAGE and transfer protocols. In immunohistochemistry and immunofluorescence microscopy, the antibody can visualize protein localization in plant tissue sections, providing valuable spatial information about protein expression patterns . Immunoprecipitation (IP) using At3g58950 Antibody can enrich the target protein and its interacting partners from complex plant extracts, facilitating subsequent mass spectrometry analysis to identify protein complexes . Chromatin immunoprecipitation (ChIP) might be possible if the protein has DNA-binding properties. Flow cytometry applications could analyze protein expression at the cellular level in plant protoplasts. Each application requires optimization of antibody concentrations, incubation conditions, and detection methods for optimal results.

What experimental controls should be included when using At3g58950 Antibody?

Rigorous experimental design with appropriate controls is critical when using At3g58950 Antibody. Include a positive control using known positive plant material (Arabidopsis thaliana tissue expressing At3g58950) alongside experimental samples to verify antibody functionality . Incorporate negative controls including: (1) omission of primary antibody to assess secondary antibody specificity, (2) pre-immune serum at equivalent concentration to evaluate background binding, and (3) competing peptide controls where the antibody is pre-incubated with immunizing peptide to demonstrate binding specificity . For tissue-specific studies, include tissues known to express and not express the target protein based on transcriptomic data. When possible, utilize genetic controls such as knockout/knockdown lines or overexpression lines to validate antibody specificity. For quantitative applications, include standard curves with purified recombinant protein where feasible. Implementing these controls will enhance data reliability and facilitate accurate interpretation of experimental results.

How can At3g58950 Antibody be optimized for immunofluorescence microscopy in plant tissues?

Optimizing At3g58950 Antibody for immunofluorescence microscopy in plant tissues requires careful method development. Begin with fixation optimization, testing both cross-linking (paraformaldehyde) and precipitating (acetone/methanol) fixatives to determine which best preserves antigen epitopes while maintaining tissue morphology . Antigen retrieval methods may be necessary for paraffin-embedded sections; test citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0) at different temperatures. For cell wall penetration, include appropriate permeabilization steps using detergents like Triton X-100 or enzymatic digestion with cellulase/macerozyme. Blocking solutions should be optimized to reduce background; test BSA, normal serum from the secondary antibody host species, or commercial blocking reagents. Perform antibody dilution series (typically 1:100 to 1:1000) to determine optimal concentration balancing specific signal and background. Incubation parameters (time, temperature) should be systematically tested. Include appropriate controls as described in 2.3, and counterstain with DAPI to visualize nuclei and provide cellular context to protein localization patterns.

How does At3g58950 protein interact with other components of plant cellular machinery?

Understanding At3g58950 protein interactions requires integrated experimental approaches. As an F-box/RNI-like superfamily protein, At3g58950 likely functions within SCF (Skp1-Cullin-F-box) ubiquitin ligase complexes . Researchers can employ co-immunoprecipitation (co-IP) using At3g58950 Antibody followed by mass spectrometry analysis to identify interacting proteins in plant extracts . Yeast two-hybrid screens can complement these findings by detecting direct protein-protein interactions. Bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer (FRET) microscopy allow visualization of protein interactions in planta. For functional studies, researchers should investigate the effects of At3g58950 mutation or overexpression on potential substrate protein levels, particularly during developmental transitions or stress responses where F-box proteins often play regulatory roles. Comparative transcriptomics and proteomics between wild-type and At3g58950 mutant plants can reveal downstream pathways affected by At3g58950 activity. Integration of these datasets will provide a comprehensive view of At3g58950's role within plant cellular networks and identify key interaction partners for further investigation.

What approaches can be used to study At3g58950 protein internalization and trafficking?

Studying At3g58950 protein internalization and trafficking requires specialized techniques adapted from established antibody internalization assays. Real-time visualization using fluorescently labeled At3g58950 Antibody can track protein movement within living plant cells . Researchers can employ pH-sensitive fluorescent dyes like Incucyte® Fabfluor-pH to specifically label the antibody, allowing detection of internalization events as pH changes occur during vesicular trafficking . Time-lapse imaging captures the dynamics of protein movement, with normalized red object area measurements providing quantitative data on internalization rates . Cell density titration experiments are essential to determine optimal conditions, as internalization responses typically show cell number dependence . For fixed-cell approaches, researchers can use immunogold labeling with At3g58950 Antibody for transmission electron microscopy to precisely localize the protein within cellular compartments. Pharmacological inhibitors of specific trafficking pathways (e.g., brefeldin A for Golgi-mediated transport) can identify the mechanisms involved in At3g58950 trafficking. These approaches collectively provide insights into the dynamic behavior of At3g58950 protein in cellular contexts.

How can active learning approaches improve At3g58950 antibody-antigen binding prediction?

Active learning strategies can significantly enhance antibody-antigen binding prediction for At3g58950 research, reducing experimental costs and accelerating discovery. This approach begins with a small labeled dataset of At3g58950 antibody-antigen interactions and iteratively expands it based on machine learning prediction uncertainty . Compared to random data labeling, optimized active learning algorithms have demonstrated up to 35% reduction in required antigen mutant variants and 28-step acceleration in the learning process . For At3g58950 Antibody research, this translates to more efficient epitope mapping and binding optimization. Implementation involves: (1) generating an initial small dataset of verified At3g58950 antibody-antigen binding pairs, (2) training preliminary machine learning models on this data, (3) using model uncertainty to identify informative new samples for experimental testing, and (4) iteratively retraining models with expanded datasets . This process efficiently builds a comprehensive binding landscape for At3g58950 Antibody, enabling researchers to predict interactions with mutated forms of the protein or potential cross-reactive targets. This approach is particularly valuable for out-of-distribution predictions where test antibodies and antigens differ from training data.

What strategies can identify specific epitopes recognized by At3g58950 Antibody?

Epitope mapping for At3g58950 Antibody provides crucial information for experimental design and interpretation. Researchers can employ peptide array analysis using overlapping synthetic peptides spanning the At3g58950 protein sequence immobilized on solid support. The antibody is then applied to the array, and binding is detected using labeled secondary antibodies. Regions showing strong binding signal represent potential linear epitopes. For conformational epitopes, hydrogen-deuterium exchange mass spectrometry (HDX-MS) can identify regions protected from deuterium incorporation when the antibody is bound to the protein. X-ray crystallography or cryo-electron microscopy of the antibody-antigen complex provides the highest resolution view of specific binding interactions, though these methods are resource-intensive. Computational approaches including epitope prediction algorithms and molecular docking can complement experimental data. Alanine scanning mutagenesis, where amino acids in the suspected epitope region are systematically replaced with alanine, can identify critical residues for antibody recognition. Understanding the specific epitopes recognized by At3g58950 Antibody enables more precise experimental design and interpretation of results, particularly when studying protein variants or closely related family members.

How can heterophilic antibody interference be addressed in At3g58950 immunoassays?

Heterophilic antibodies in plant or animal samples can form immune complexes with research antibodies, resulting in false signals and reduced analyte recognition . When using At3g58950 Antibody in two-site immunoassays, researchers should implement multiple strategies to minimize this interference. First, incorporate blocking agents specific for heterophilic antibodies in assay buffers, such as non-immune serum from the same species as the detection antibody, commercial heterophilic blocking reagents, or polymerized IgG. Second, design assay formats that reduce heterophilic antibody binding opportunities, such as using F(ab')2 fragments instead of whole IgG molecules. Third, validate results with orthogonal methods that are less susceptible to heterophilic interference. If interference is detected, implement serial dilution analysis to distinguish true from false positive results, as heterophilic interference typically does not follow the expected dilution pattern. When reporting results, document all measures taken to address potential heterophilic antibody interference. For quantitative assays, consider generating a correction factor by comparing results before and after heterophilic antibody removal or blocking steps.

What approaches can resolve weak or inconsistent signals when using At3g58950 Antibody?

When encountering weak or inconsistent signals with At3g58950 Antibody, a systematic troubleshooting approach is essential. First, verify antibody quality with a dot blot of purified antigen at different concentrations. For Western blotting applications, optimize protein extraction by testing different extraction buffers to ensure efficient solubilization of membrane-associated F-box proteins, and include protease inhibitors to prevent target degradation. Adjust protein loading amounts (5-50 μg per lane) and antibody concentration (1:100 to 1:5000 dilutions). Extend primary antibody incubation time or temperature (overnight at 4°C versus 1-2 hours at room temperature). For immunohistochemistry, test different fixation methods and antigen retrieval protocols as overfixation can mask epitopes. Enhance detection sensitivity by employing signal amplification systems like tyramide signal amplification or polymer-based detection systems. Consider tissue-specific expression patterns of At3g58950, as the protein may be expressed at different levels across tissues or developmental stages . Document all optimization steps and maintain consistent protocols once optimized to ensure reproducibility across experiments.

How should researchers evaluate batch-to-batch variation in At3g58950 Antibody?

Consistent antibody performance across experiments requires systematic evaluation of batch-to-batch variation. Upon receiving a new batch of At3g58950 Antibody, researchers should perform comparative analysis with the previous batch using identical protocols and samples. Begin with a titration experiment using serial dilutions (e.g., 1:100, 1:500, 1:1000, 1:5000) of both antibody batches to determine optimal working concentrations and compare signal intensity curves. Western blot analysis using standardized Arabidopsis protein samples should evaluate specificity by comparing banding patterns between batches. Calculate signal-to-noise ratios for quantitative comparison. For immunohistochemistry applications, perform side-by-side staining of serial tissue sections to assess staining patterns and intensities. Establish internal reference standards, such as recombinant At3g58950 protein at known concentrations, to normalize signals across batches. Maintain detailed records of batch comparison results in a laboratory information management system, including lot numbers, receipt dates, and performance metrics. If significant variation is observed, adjust protocols accordingly and note the changes in experimental documentation to maintain data comparability across studies.

What strategies can prevent non-specific binding in At3g58950 Antibody experiments?

Non-specific binding can compromise experimental results with At3g58950 Antibody, but several strategies can mitigate this issue. Optimize blocking conditions by testing different blocking agents (BSA, non-fat dry milk, normal serum, commercial blocking solutions) at various concentrations (1-5%) and incubation times (30 minutes to overnight). For plant tissue experiments, include plant-specific blocking reagents such as powdered plant material from non-target species. Include detergents like Tween-20 (0.05-0.1%) in washing and antibody diluent buffers to reduce hydrophobic interactions. Pre-absorb the antibody with acetone powder prepared from tissues lacking the target protein to remove antibodies that recognize common plant antigens. For immunohistochemistry, autofluorescence can be mistaken for specific signal; include unstained controls and consider treatment with sodium borohydride or Sudan Black B to reduce plant tissue autofluorescence. Titrate both primary and secondary antibodies to determine minimum concentrations giving specific signals. In Western blots, transfer efficiency and membrane blocking should be optimized; consider alternative membrane types (PVDF versus nitrocellulose) if non-specific binding persists. Document successful protocols thoroughly to ensure reproducibility across experiments.

How is At3g58950 Antibody being used in plant developmental studies?

At3g58950 Antibody serves as a valuable molecular marker in plant developmental biology, particularly in floral organ development research. Immunofluorescence microscopy with this antibody has revealed specific protein localization patterns in Arabidopsis inflorescence tissue sections, with expression observed in particular cell layers . This spatial information provides insights into potential functions during flower development. Researchers are employing At3g58950 Antibody in time-course studies to track protein expression throughout developmental stages, correlating protein levels with specific developmental events. The antibody is also being used in comparative studies across wild-type and mutant plants to assess how developmental abnormalities correlate with At3g58950 protein expression or localization changes. Integration with other molecular markers allows multicolor imaging to examine spatial relationships between At3g58950 and other developmentally regulated proteins. As an F-box protein family member, At3g58950 may participate in protein degradation pathways critical for developmental transitions; the antibody enables researchers to investigate these regulatory networks by identifying co-localized proteins and tracking expression dynamics during key developmental events.

What high-throughput screening approaches can enhance At3g58950 Antibody research?

High-throughput screening methodologies significantly accelerate At3g58950 research by enabling rapid, parallel analysis of multiple experimental conditions. Researchers can adapt At3g58950 Antibody for 96-well or 384-well format assays similar to established antibody internalization protocols that demonstrate Z' values exceeding 0.75, indicating excellent assay robustness . This approach facilitates concurrent testing of diverse variables including protein expression across multiple plant tissues, developmental stages, or stress conditions. Automated image acquisition and analysis systems, like the Incucyte® platform, enable real-time monitoring of protein dynamics in response to experimental treatments . For epitope mapping and cross-reactivity studies, peptide microarrays with overlapping peptides from At3g58950 and related proteins can be probed simultaneously. Library-on-library screening approaches allow testing At3g58950 Antibody against numerous protein variants to characterize binding specificity and identify critical interaction residues . The incorporation of machine learning algorithms can optimize experimental design by predicting which conditions or protein variants would be most informative for testing . These high-throughput approaches generate comprehensive datasets that reveal patterns not discernible from limited experimental conditions, accelerating discovery while reducing resource requirements.

What are the emerging applications of At3g58950 Antibody in plant stress response research?

The At3g58950 Antibody is increasingly utilized to investigate plant stress responses, where F-box proteins often play crucial regulatory roles. Researchers are employing immunoprecipitation followed by mass spectrometry (IP-MS) to identify stress-responsive proteins that interact with At3g58950 under various environmental challenges . These interaction networks provide insights into how protein degradation pathways reconfigure during stress adaptation. Comparative immunohistochemistry between control and stressed plants reveals changes in At3g58950 protein localization or abundance, indicating potential stress-specific functions. Time-course experiments track dynamic changes in protein expression following stress exposure, helping establish the temporal sequence of stress response pathways. Western blot analysis quantifies protein level changes across different stress intensities and durations, enabling correlation with physiological responses. Researchers are also investigating post-translational modifications of At3g58950 under stress conditions, as these often regulate F-box protein activity and substrate specificity. The integration of these antibody-based approaches with transcriptomic and metabolomic data provides a comprehensive view of how At3g58950 contributes to plant stress resilience, potentially informing strategies for developing more stress-tolerant crops.