At4g35120 Antibody

What is the At4g35120 protein and why is it significant in Arabidopsis research?

At4g35120 (O49618) is a protein encoded by the Arabidopsis thaliana genome, specifically located on chromosome 4. This protein is significant in plant biology research because it serves as a model for understanding plant development, stress responses, and cellular processes. The At4g35120 gene encodes a specific protein that has been implicated in various physiological and developmental pathways in plants. Understanding its function through antibody-based detection helps researchers investigate its role in plant growth, development, and response to environmental stressors. The protein's conservation across various plant species makes it valuable for comparative studies in plant molecular biology and genetics. Researchers often use At4g35120 antibodies to track protein expression, localization, and interaction with other cellular components in response to different experimental conditions .

How do I design a basic western blot experiment using At4g35120 antibody?

When designing a western blot experiment using At4g35120 antibody, start by preparing protein extracts from Arabidopsis tissues using an appropriate extraction buffer that preserves protein integrity while minimizing degradation. The experimental design should include proper controls such as positive controls (purified At4g35120 protein if available), negative controls (tissues where the protein is not expressed), and loading controls (housekeeping proteins like actin or tubulin) to ensure reliable interpretation of results . For protein separation, use SDS-PAGE with an appropriate percentage acrylamide gel based on the expected molecular weight of At4g35120. After transfer to a membrane, block with 5% non-fat dry milk or BSA in TBST before incubating with the primary At4g35120 antibody at the recommended dilution (typically 1:1000 to 1:5000) overnight at 4°C. The experimental protocol should include thorough washing steps between antibody applications to reduce background noise and increase signal specificity, followed by visualization using an appropriate detection system based on your laboratory's equipment .

What are the optimal storage conditions for maintaining At4g35120 antibody activity?

The optimal storage conditions for maintaining At4g35120 antibody activity include storing the antibody at -20°C for long-term storage and at 4°C for antibodies in current use, with minimal freeze-thaw cycles to prevent degradation of the antibody protein structure. Most polyclonal antibodies targeting plant proteins like At4g35120 remain stable for at least 12-24 months when properly stored, though activity should be monitored periodically using positive controls to ensure consistent performance in experimental applications. The antibody should be aliquoted into smaller volumes upon receipt to minimize repeated freeze-thaw cycles, which can significantly reduce antibody effectiveness over time. When handling the antibody, avoid contamination by using sterile techniques and appropriate personal protective equipment to prevent the introduction of proteases or other contaminants that could degrade the antibody . For working solutions, store at 4°C and use within 2-4 weeks, as diluted antibodies typically have reduced stability compared to concentrated stock solutions.

How can I optimize immunoprecipitation protocols for At4g35120 protein complex isolation?

Optimizing immunoprecipitation protocols for At4g35120 protein complex isolation requires careful consideration of lysis buffer composition, antibody binding conditions, and washing stringency to maintain native protein-protein interactions while minimizing non-specific binding. Begin by testing different cell lysis buffers (varying detergent types and concentrations) to identify conditions that solubilize At4g35120 effectively while preserving its interactions with binding partners. For plant tissues, consider buffers containing 0.5-1% NP-40 or Triton X-100, supplemented with protease inhibitor cocktails and phosphatase inhibitors if studying phosphorylation-dependent interactions . Pre-clear lysates with protein A/G beads to remove components that bind non-specifically to the beads before adding the At4g35120 antibody. Experiment with antibody-to-lysate ratios and incubation times (typically 2-4 hours at 4°C or overnight) to determine optimal conditions for efficient target capture without overwhelming the system with antibody. Implement a stepped washing protocol with decreasing stringency to remove non-specific binders while retaining true interacting partners, and validate results using reciprocal co-immunoprecipitation or proximity labeling approaches to confirm the biological relevance of identified interactions .

What are the critical considerations when designing ChIP experiments using At4g35120 antibody?

When designing Chromatin Immunoprecipitation (ChIP) experiments with At4g35120 antibody, researchers must optimize several critical parameters to ensure meaningful results about protein-DNA interactions. First, proper crosslinking conditions are essential—typically 1% formaldehyde for 10-15 minutes for plant tissues, but this may require optimization for specific tissues to achieve sufficient crosslinking without overfixation that could mask epitopes or hinder chromatin shearing . Sonication parameters must be carefully established to generate chromatin fragments of appropriate size (typically 200-500 bp), which requires empirical testing with different sonication times and amplitudes followed by agarose gel analysis of fragment distribution. Antibody specificity is particularly crucial for ChIP applications, as non-specific binding can lead to false positive genomic targets; therefore, validation through western blotting of nuclear extracts and inclusion of appropriate controls (such as IgG control, input samples, and known positive/negative target regions) is imperative . Consider implementing spike-in controls with chromatin from a different species to normalize for technical variability between samples, especially when comparing treatments that might affect global chromatin accessibility. For low-abundance transcription factors or chromatin-associated proteins like those potentially interacting with At4g35120, techniques such as ChIP-exo or CUT&RUN may provide higher resolution and sensitivity compared to standard ChIP-seq approaches .

How can I troubleshoot weak or absent signals when using At4g35120 antibody in immunofluorescence studies?

Troubleshooting weak or absent signals in immunofluorescence studies with At4g35120 antibody requires systematic evaluation and optimization of each experimental step from fixation to imaging. Begin by assessing fixation protocols, as overfixation can mask epitopes while underfixation may lead to poor morphology preservation; test multiple fixatives (4% paraformaldehyde, methanol, or combined protocols) and fixation times to determine optimal conditions for epitope accessibility . Evaluate permeabilization methods, as inadequate permeabilization prevents antibody access to intracellular antigens; test different detergents (Triton X-100, Tween-20, or saponin) at various concentrations and durations to optimize membrane permeabilization without disrupting cellular structures. Implement antigen retrieval techniques such as heat-induced epitope retrieval (HIER) or enzymatic retrieval if the target epitope might be masked due to protein crosslinking or conformational changes during fixation. Optimize antibody concentration through titration experiments, testing a range of dilutions to find the optimal signal-to-noise ratio, and consider extended incubation times (overnight at 4°C) to enhance specific binding . Evaluate detection systems by comparing different secondary antibodies, fluorophores with appropriate spectral properties, and signal amplification methods like tyramide signal amplification (TSA) for low-abundance proteins, while ensuring proper microscope settings with appropriate filter sets and exposure times to capture specific signals without introducing artifacts .

What approaches can be used to study post-translational modifications of At4g35120 protein?

To study post-translational modifications (PTMs) of At4g35120 protein, researchers should employ integrated approaches combining enrichment strategies, high-resolution mass spectrometry, and functional validation techniques. Start with phosphorylation analysis using phospho-specific antibodies against known or predicted phosphorylation sites on At4g35120, complemented by phospho-enrichment techniques such as immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO₂) chromatography prior to mass spectrometry analysis . For ubiquitination studies, employ tandem ubiquitin binding entities (TUBEs) or antibodies specific to ubiquitin remnants after trypsin digestion (K-ε-GG) for enrichment, followed by mass spectrometry to identify ubiquitinated lysine residues. Implement site-directed mutagenesis to generate point mutations at putative modification sites, creating non-modifiable variants (e.g., serine to alanine for phosphorylation sites) to assess the functional significance of specific modifications in vivo. Consider using chemical inhibitors or genetic approaches (kinase/phosphatase mutants) to modulate the cellular machinery responsible for specific modifications and observe the consequent effects on At4g35120 function or localization . For temporal dynamics of modifications, employ pulse-chase experiments with metabolic labeling or time-course studies following stimulus application, combined with selected reaction monitoring (SRM) mass spectrometry to quantify specific modified peptides with high sensitivity and reproducibility across multiple samples .

How should I design proper controls for At4g35120 antibody experiments in different plant tissues?

Designing proper controls for At4g35120 antibody experiments across different plant tissues requires a multilayered approach to account for tissue-specific variations in protein expression, matrix effects, and potential cross-reactivity. Always include positive control tissues where At4g35120 is known to be expressed based on transcriptomic data or previous studies, as well as negative control tissues from knockout/knockdown plants or tissues known to lack expression of the target protein . Incorporate technical controls including secondary antibody-only controls to assess non-specific binding of the detection system, isotype controls using non-specific antibodies of the same isotype to evaluate background binding, and pre-immune serum controls if using polyclonal antibodies to establish baseline reactivity before immunization. For cross-species applications, validate antibody specificity in each species through western blotting before proceeding with more complex applications like immunohistochemistry or immunoprecipitation . Implement loading controls appropriate for each tissue type, recognizing that traditional housekeeping proteins may show tissue-specific variations in expression; consider multiple loading controls or total protein staining methods like Ponceau S to normalize for loading differences. When comparing protein expression across developmental stages or stress conditions, include internal reference samples processed in parallel across all experimental batches to control for technical variation in antibody performance or detection sensitivity .

What statistical approaches are recommended for analyzing quantitative data from At4g35120 antibody experiments?

When analyzing quantitative data from At4g35120 antibody experiments, researchers should implement robust statistical approaches that account for the specific characteristics and limitations of immunological detection methods. Begin with exploratory data analysis, including normality testing (Shapiro-Wilk test) and variance homogeneity assessment (Levene's test) to determine whether parametric or non-parametric statistical methods are appropriate for your dataset . For western blot densitometry analysis, employ technical replicates (multiple lanes of the same sample) and biological replicates (independent samples) to estimate technical and biological variability, respectively, and use appropriate normalization methods (typically to loading controls or total protein) to account for loading differences. When comparing multiple experimental conditions, use ANOVA followed by appropriate post-hoc tests (such as Tukey's HSD for balanced designs or Games-Howell for unequal variances) rather than multiple t-tests to control family-wise error rate. For immunofluorescence quantification, consider cell-to-cell variability by measuring multiple cells per sample and implement hierarchical statistical models that account for the nested structure of the data (cells within samples within treatments) . Implement power analysis before designing experiments to determine appropriate sample sizes, recognizing that antibody-based methods often show higher variability than other quantitative techniques, requiring larger sample sizes to achieve sufficient statistical power. For complex experimental designs with multiple factors or repeated measures, consider mixed-effects models that can account for random effects (such as batch variation) while testing for fixed effects of experimental treatments .

How can I address potential cross-reactivity issues with At4g35120 antibody in closely related plant species?

Addressing potential cross-reactivity issues with At4g35120 antibody when working with closely related plant species requires comprehensive validation strategies and careful experimental design. Begin by performing sequence alignment analysis of the immunizing peptide or protein region across species of interest to identify regions of high conservation or divergence, which can predict potential cross-reactivity or specificity issues . Validate antibody specificity in each species through western blotting, looking for single bands of the expected molecular weight and comparing band patterns between species to identify potential cross-reactive proteins. Implement peptide competition assays in each species separately, using the immunizing peptide to block specific binding sites on the antibody, which should eliminate specific signals but not non-specific cross-reactivity . Consider using heterologous expression systems to express the target protein from different species with epitope tags, allowing direct comparison of antibody recognition efficiency across orthologs. For critical applications, complement antibody-based detection with orthogonal techniques such as mass spectrometry or activity-based protein profiling to confirm protein identity. In comparative studies, implement consistent extraction and detection protocols across all species to minimize technical variables that could be misinterpreted as biological differences, and include appropriate positive and negative controls for each species rather than extrapolating control results from a single species .

What are the best practices for determining the optimal antibody concentration for different experimental applications?

Determining the optimal antibody concentration for different experimental applications requires systematic titration approaches tailored to each specific technique's sensitivity and dynamic range. For western blotting, perform an antibody titration series (typically ranging from 1:500 to 1:10,000) using consistent protein amounts across all conditions, and select the concentration that provides clear specific bands with minimal background; this approach balances signal intensity with reagent conservation and specificity . In immunohistochemistry or immunofluorescence applications, titrate antibody concentrations against fixed tissue samples known to express At4g35120, comparing signal-to-background ratios across concentrations while maintaining consistent incubation times and detection methods. For immunoprecipitation experiments, optimize antibody-to-lysate ratios rather than simply focusing on antibody concentration, testing different amounts of antibody against constant lysate volumes to identify conditions that maximize target capture without introducing excess antibody that could contribute to non-specific binding . In flow cytometry applications, calculate the antibody saturation point by titrating antibody concentration and plotting the median fluorescence intensity against antibody concentration, then selecting a concentration on the plateau of the resulting curve. Recognize that optimal concentrations may differ significantly between applications (e.g., western blotting typically requires lower concentrations than immunohistochemistry) and between sample types (e.g., different tissues or fixation methods), necessitating separate optimization for each experimental context .

How can I use At4g35120 antibody in multiplexed immunoassays with other Arabidopsis proteins?

Developing multiplexed immunoassays with At4g35120 antibody and other Arabidopsis protein antibodies requires careful planning to avoid cross-reactivity, signal interference, and detection limitations. Begin by selecting antibodies raised in different host species (e.g., rabbit anti-At4g35120 paired with mouse anti-AGO4) to enable simultaneous detection using species-specific secondary antibodies conjugated to distinct fluorophores or enzymes . Verify antibody compatibility by testing each antibody individually and in combination on the same samples to ensure that performance is not compromised when used together. For fluorescence-based multiplexing, select fluorophores with minimal spectral overlap and implement appropriate controls for spectral compensation if using flow cytometry or confocal microscopy with multiple fluorescence channels. Consider sequential immunostaining protocols when using multiple antibodies from the same host species, which involves complete detection and blocking of the first primary antibody before applying the second, though this approach may introduce additional variables and potential artifacts . For protein co-localization studies, implement quantitative colocalization analysis using appropriate metrics (such as Manders' or Pearson's correlation coefficients) rather than relying on visual assessment of overlay images. In cases where traditional immunostaining approaches are limiting, consider implementing cyclic immunofluorescence or mass cytometry (CyTOF) for highly multiplexed protein detection, though these advanced techniques require specialized equipment and extensive optimization .

What strategies can resolve inconsistent results when using At4g35120 antibody across different plant growth conditions?

Resolving inconsistent results when using At4g35120 antibody across different plant growth conditions requires systematic troubleshooting of both biological and technical variables that might influence antibody performance or protein expression. First, standardize sample collection timing and procedures, as protein expression and modification states can vary significantly with circadian rhythms, developmental stages, and even handling stress responses in plants . Implement comprehensive extraction buffer optimization for each condition, as different growth conditions may alter tissue composition, requiring adjustments to buffer components (detergents, salt concentration, pH) to maintain consistent protein extraction efficiency. Consider that growth conditions may alter post-translational modifications or protein complex formation, potentially masking or exposing antibody epitopes; test multiple antibodies targeting different regions of At4g35120 if available, or use denaturing conditions that may normalize epitope accessibility . Establish rigorous normalization strategies—beyond standard loading controls—such as spike-in standards or absolute quantification approaches when comparing across drastically different growth conditions where traditional housekeeping proteins might be differentially regulated. Implement batch controls by processing samples from different conditions in parallel and including reference samples in each experiment to distinguish technical variability from true biological effects. Document all experimental variables meticulously, including growth chamber specifications, light intensity and photoperiod, watering regimes, and any treatments applied, as seemingly minor variations in growth conditions can significantly impact protein expression patterns or extraction efficiency .

How can I adapt At4g35120 antibody protocols for high-throughput screening applications?

Adapting At4g35120 antibody protocols for high-throughput screening applications requires optimizing for automation compatibility, miniaturization, and robust quality control while maintaining assay sensitivity and specificity. Begin by transitioning from traditional western blotting to microplate-based immunoassays such as ELISA or AlphaLISA, which offer greater throughput capacity and quantitative precision for protein detection across many samples . Optimize sample preparation by developing streamlined extraction protocols compatible with multi-channel pipetting or liquid handling robots, potentially using 96-well plate-based homogenization methods and filtration plates for clarification rather than centrifugation steps. Implement automated liquid handling systems for consistent antibody dilution, sample dispensing, and washing steps to minimize pipetting errors and reduce assay variability across plates. Develop quality control metrics including Z′-factor calculations to assess assay robustness, implement plate layout strategies that control for position effects (edge effects), and include standard curves and reference samples on each plate to enable cross-plate normalization . Consider bead-based multiplexing technologies like Luminex, which allow simultaneous detection of multiple proteins in a single well when combining At4g35120 antibody with other target antibodies of interest. Establish clear criteria for hit identification and validation, including statistical thresholds appropriate for the scale of screening and secondary confirmation assays to validate primary hits, recognizing that high-throughput approaches typically involve trade-offs between throughput and depth of characterization .

What are the most effective approaches for studying low-abundance At4g35120 protein in specific cell types?

Studying low-abundance At4g35120 protein in specific cell types requires specialized techniques that enhance detection sensitivity while maintaining cellular and spatial context. Implement laser capture microdissection to isolate specific cell types of interest before protein extraction, enabling enrichment of target cells and removal of surrounding tissues that might dilute the signal from rare cell populations . Consider proximity ligation assays (PLA) which provide signal amplification through rolling circle amplification while maintaining spatial resolution, allowing detection of individual protein molecules or protein complexes containing At4g35120 within specific cellular contexts. Utilize translating ribosome affinity purification (TRAP) technology by generating transgenic plants expressing a tagged ribosomal protein under a cell-type-specific promoter, allowing isolation of actively translating mRNAs and associated nascent proteins including At4g35120 from specific cell types . For immunohistochemical applications, implement tyramide signal amplification (TSA) or other enzymatic amplification methods that can enhance detection sensitivity by 10-100 fold compared to conventional methods while maintaining spatial resolution. Consider cell-type-specific expression of epitope-tagged versions of At4g35120 under its native promoter using CRISPR-based knock-in strategies, allowing detection with highly specific anti-tag antibodies that often offer superior sensitivity compared to antibodies against the native protein . Complement antibody-based approaches with highly sensitive mass spectrometry techniques such as selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) targeting specific peptides from At4g35120, which can achieve femtomole-level detection limits from small amounts of starting material .

How can I effectively combine At4g35120 antibody-based techniques with transcriptomic data analysis?

Effectively combining At4g35120 antibody-based protein detection with transcriptomic data requires integrative approaches that account for the different regulatory levels and temporal dynamics of RNA and protein expression. Begin by designing experiments with matched samples for both transcriptomic (RNA-seq, microarray) and proteomic (western blot, immunoprecipitation) analyses to enable direct correlation between mRNA and protein levels across conditions or treatments . Implement time-course studies that capture both immediate transcriptional responses and the delayed protein-level changes that follow, recognizing that protein abundance often lags behind transcript changes by hours to days in plant systems. Distinguish between correlation and causation by complementing observational studies with perturbation experiments, such as using inducible expression systems to manipulate At4g35120 levels and observe consequent changes in both the transcriptome and proteome . Utilize computational approaches including regression analysis to model the relationship between transcript and protein levels, accounting for factors such as translation efficiency, protein half-life, and post-translational regulation that may explain discrepancies between transcriptomic and proteomic data. For single-cell or tissue-specific analyses, combine single-cell RNA-seq with antibody-based techniques like immunohistochemistry or flow cytometry on parallel samples to map the spatial distribution of At4g35120 expression across different cell types or tissues identified in the transcriptomic data . Implement network analysis approaches that integrate transcriptomic data with protein-protein interaction data (from co-immunoprecipitation or yeast two-hybrid screens) to place At4g35120 within functional pathways and regulatory networks that explain its biological role .

What are the best practices for integrating At4g35120 antibody results with proteomics data?

Integrating At4g35120 antibody results with proteomics data requires careful experimental design and analytical approaches that leverage the complementary strengths of targeted antibody-based detection and unbiased mass spectrometry-based proteomics. Design experiments to include samples for both antibody-based methods and mass spectrometry analysis, ideally using identical or split samples to minimize biological variability when comparing results across platforms . Use antibody-based immunoprecipitation followed by mass spectrometry (IP-MS) to identify interaction partners of At4g35120, providing a focused protein interaction network that can be contextualized within the broader proteomic landscape. Implement targeted proteomics approaches such as parallel reaction monitoring (PRM) or selected reaction monitoring (SRM) to quantify specific peptides from At4g35120 and its interactors with high sensitivity, serving as orthogonal validation of antibody-based quantification . For discrepancies between antibody-based quantification and proteomics data, investigate potential explanations including post-translational modifications, protein complex formation, or isoform-specific expression that might differentially affect antibody recognition versus peptide detection in mass spectrometry. Create integrative visualizations that combine quantitative antibody-based results with proteomic data, such as overlaying western blot quantification on volcano plots from differential proteomics or mapping antibody-validated interactions onto broader protein-protein interaction networks . Consider the dynamic range limitations of each technique—antibody-based methods often having greater sensitivity for low-abundance proteins while shotgun proteomics provides broader coverage—and design experiments that leverage these complementary strengths rather than expecting perfect concordance between approaches .

How can metabolomic analyses complement At4g35120 antibody-based functional studies?

Integrating metabolomic analyses with At4g35120 antibody-based functional studies can provide comprehensive insights into the protein's role in metabolic networks and cellular physiology. Design parallel experiments that simultaneously collect samples for metabolite extraction and protein analysis, allowing direct correlation between At4g35120 protein levels (detected via antibody-based methods) and metabolite profiles under identical conditions . Implement metabolic flux analysis using stable isotope labeling to track the flow of metabolites through pathways potentially regulated by At4g35120, complementing static protein abundance measurements with dynamic information on metabolic activity. Consider using inducible expression systems or conditional mutants to systematically manipulate At4g35120 levels or activity, then measure consequent metabolic changes to establish causal relationships rather than mere correlations . For proteins involved in metabolic regulation like many Arabidopsis proteins, examine post-translational modifications using modification-specific antibodies in parallel with metabolomics to understand how protein regulation connects to metabolic outcomes. Implement network integration approaches that combine protein interaction data (from immunoprecipitation) with metabolic correlation networks to identify potential metabolic modules under At4g35120 influence, particularly focusing on metabolites that show strong correlation with protein abundance or modification state across conditions . Consider subcellular fractionation followed by both antibody detection of At4g35120 and metabolite profiling of the same fractions to establish compartment-specific connections between protein localization and metabolic activities, which is particularly relevant for proteins that may shuttle between cellular compartments in response to metabolic status .

What approaches can be used to study protein-protein interactions involving At4g35120 protein?

Studying protein-protein interactions involving At4g35120 requires a multi-faceted approach combining in vitro, in vivo, and computational methods to comprehensively characterize the interactome. Begin with co-immunoprecipitation (co-IP) using At4g35120 antibody followed by mass spectrometry to identify interaction partners under native conditions, implementing stringent controls including IgG pulldowns and, ideally, immunoprecipitation from knockout/knockdown lines to distinguish true interactors from background binding proteins . Validate key interactions through reciprocal co-IP experiments, pulling down identified partners with their specific antibodies and blotting for At4g35120 to confirm bidirectional interaction. Implement proximity-based labeling approaches such as BioID or TurboID by fusing these promiscuous biotin ligases to At4g35120, allowing biotinylation of proteins in close proximity followed by streptavidin pulldown and mass spectrometry identification, which captures both stable and transient interactions in the native cellular environment . For direct physical interaction assessment, use in vitro techniques such as surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) with purified proteins to determine binding affinities and kinetics. Visualize protein-protein interactions in planta using techniques such as bimolecular fluorescence complementation (BiFC), Förster resonance energy transfer (FRET), or split luciferase complementation, which provide spatial information about where in the cell these interactions occur . Complement experimental approaches with computational predictions using tools that leverage structural models, domain-domain interaction databases, and co-expression patterns to predict potential interactors for experimental validation, particularly useful for prioritizing candidates from high-throughput screens .