At4g10190 Antibody

What is the At4g10190 gene and what protein does it encode?

At4g10190 is a gene locus in Arabidopsis thaliana (thale cress) that encodes a protein involved in immune response pathways. The protein contains domains that function in cell signaling during pathogen response. Understanding this gene's function is critical for researchers investigating plant immunity, as it participates in signal transduction cascades that regulate defense mechanisms against pathogens. When designing experiments with At4g10190 antibodies, researchers should consider the protein's subcellular localization, which primarily includes membrane-associated regions where immune signaling occurs. Recent studies have shown that the protein undergoes post-translational modifications in response to pathogen-associated molecular patterns (PAMPs), making it an important target for studying early immune response in plants.

How should At4g10190 antibody be validated before experimental use?

Proper validation of At4g10190 antibody is crucial for ensuring experimental reliability. Begin validation with Western blot analysis using both wild-type and At4g10190 knockout/knockdown plant tissues to confirm specificity. The expected molecular weight should be compared with theoretical predictions based on amino acid sequence. Next, perform immunoprecipitation followed by mass spectrometry to confirm the antibody pulls down At4g10190 protein specifically. For immunolocalization studies, validate with appropriate controls including pre-immune serum and peptide competition assays. Additionally, cross-reactivity should be tested against closely related proteins, especially those sharing conserved domains. Document all validation results, including antibody lot numbers, as antibody performance can vary between lots. Maintaining consistent validation protocols between research groups is essential for reproducible results in the scientific community.

What are the optimal storage and handling conditions for At4g10190 antibody?

To maintain At4g10190 antibody functionality, store the concentrated stock at -80°C in small aliquots (10-50 μL) to minimize freeze-thaw cycles, which can cause antibody degradation and reduced specificity. Working dilutions should be prepared fresh and stored at 4°C for no longer than one week. Buffer compositions significantly impact antibody stability; generally, PBS with 0.02% sodium azide and 50% glycerol is recommended for long-term storage. Avoid repeated freeze-thaw cycles, which can reduce antibody titer by approximately 10-15% per cycle. When handling, minimize exposure to direct light and maintain sterile conditions to prevent microbial contamination. Document storage conditions and freeze-thaw history for each aliquot to track potential variations in experimental results. For long-term projects, consider performing validation tests at 6-month intervals to ensure consistent antibody performance over time.

How does phosphorylation state affect At4g10190 antibody binding efficiency?

The phosphorylation state of At4g10190 protein significantly impacts antibody recognition and binding. Research indicates that At4g10190 undergoes dynamic phosphorylation at multiple serine/threonine residues during pathogen response signaling. When working with phospho-specific At4g10190 antibodies, researchers must consider timing in their experimental design, as phosphorylation events are often transient and context-dependent. To effectively study phosphorylation states, utilize phosphatase inhibitor cocktails during sample preparation to preserve phosphorylation status. Comparative assays using phospho-specific and total protein antibodies can reveal the proportion of phosphorylated protein under different conditions. Mass spectrometry analysis following immunoprecipitation can identify specific phosphorylation sites and their occupancy levels. For maximum detection efficiency with phospho-specific antibodies, researchers should optimize sample preparation protocols to include phosphatase inhibitors and maintain samples at 4°C throughout processing to prevent artificial dephosphorylation events.

What methodological approaches can resolve contradictory immunolocalization data for At4g10190?

Resolving contradictory immunolocalization data for At4g10190 requires systematic troubleshooting and methodological refinement. Begin by critically evaluating fixation protocols, as overfixation can mask epitopes while underfixation may alter subcellular architecture. Compare multiple fixation methods (paraformaldehyde, glutaraldehyde, methanol) at varying concentrations and durations. Next, implement epitope retrieval techniques, including heat-induced or enzymatic methods, to potentially expose masked epitopes. Different antibody clones targeting distinct epitopes should be employed, as protein conformation and interaction partners may obscure specific regions. Super-resolution microscopy techniques (STED, PALM, STORM) provide enhanced spatial resolution that may clarify ambiguous localization patterns. Corroborate immunolocalization findings with complementary approaches such as subcellular fractionation followed by Western blotting, or expression of fluorescently-tagged At4g10190 in living cells. Lastly, consider developmental and environmental influences on protein localization, as At4g10190 may shuttle between compartments under different conditions or developmental stages.

How can researchers distinguish between specific and non-specific binding when using At4g10190 antibody in complex plant tissue samples?

Distinguishing specific from non-specific binding is particularly challenging in complex plant tissues due to secondary metabolites and cell wall components that can interfere with antibody interactions. Implement a multi-layered validation approach beginning with genetic controls (knockout/knockdown lines) alongside wild-type samples processed identically. Pre-adsorption controls, where the antibody is pre-incubated with excess purified antigen before application to samples, should abolish specific signals while leaving non-specific binding intact. Titrate antibody concentrations to determine the optimal signal-to-noise ratio, as excessive antibody concentrations increase non-specific binding. Include multiple blocking agents (BSA, normal serum, plant-derived proteins) to identify the most effective blocking strategy for your specific tissue type. Secondary antibody-only controls are essential to identify background fluorescence or non-specific binding of secondary antibodies. For quantitative applications, implement computational approaches to establish signal threshold values based on control samples. Lastly, cross-validate findings using alternative detection methods such as RNA in situ hybridization or promoter-reporter fusions to confirm expression patterns.

What is the optimal protein extraction protocol for detecting At4g10190 in different plant tissues?

The optimal protein extraction protocol for At4g10190 detection varies depending on tissue type and downstream applications. For general purposes, a RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors yields good results for soluble protein fractions. For membrane-associated forms of At4g10190, incorporate 1% Triton X-100 or 0.5% CHAPS to enhance solubilization. When extracting from tissues with high phenolic content, include 2% PVPP and 5 mM ascorbic acid to prevent protein modification by oxidized phenolics. Tissue disruption should be performed rapidly at 4°C using a bead beater for young tissues or cryogenic grinding for fibrous tissues. Following extraction, centrifuge at 15,000 × g for 15 minutes at 4°C to remove cell debris. For enrichment of low-abundance At4g10190, consider implementing immunoprecipitation before Western blotting. The table below summarizes extraction buffer compositions optimized for different plant tissues:

How should researchers design co-immunoprecipitation experiments to identify interaction partners of At4g10190?

Designing effective co-immunoprecipitation (Co-IP) experiments for At4g10190 requires careful consideration of buffer conditions, crosslinking strategies, and controls. Begin with a mild lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 5% glycerol) to preserve protein-protein interactions. For transient interactions, implement in vivo crosslinking using 1% formaldehyde prior to tissue harvesting, or membrane-permeable crosslinkers such as DSP (dithiobis[succinimidyl propionate]) at 1-2 mM. Maintain all samples at 4°C throughout processing to prevent dissociation of complexes. Pre-clear lysates with Protein A/G beads to reduce non-specific binding. For the IP step, use At4g10190 antibody at a 1:50 to 1:100 dilution and incubate overnight at 4°C with gentle rotation. Include multiple controls: (1) non-immune IgG from the same species as the primary antibody, (2) lysate from knockout/knockdown plants, and (3) a competitive peptide control. After immunoprecipitation, wash beads at least four times with decreasing salt concentrations to remove non-specific binders while preserving specific interactions. For protein identification, perform mass spectrometry analysis using both label-free quantification and comparison to control IPs to distinguish specific from non-specific interactors. Validate key interactions through reciprocal Co-IP or alternative techniques such as proximity ligation assay or FRET.

What are the best practices for quantitative analysis of At4g10190 expression using immunoblotting?

Accurate quantitative analysis of At4g10190 expression via immunoblotting requires rigorous standardization and appropriate controls. Begin by determining the linear detection range for At4g10190 using a standard curve of recombinant protein or serial dilutions of a positive control sample. Load equal amounts of total protein (15-30 μg) as verified by Ponceau S staining or total protein normalization methods, which are more reliable than single housekeeping gene controls in plants. Implement technical triplicates for each biological replicate to account for transfer and detection variability. For detection, fluorescent secondary antibodies provide superior quantitative linearity compared to chemiluminescence. When quantifying bands, use integrated density values rather than peak intensity, and subtract local background values for each lane. Normalization should be performed against total protein signal (obtained from stain-free gels or Ponceau S imaging) rather than single reference proteins, especially when comparing different tissues or stress conditions that may affect reference gene expression. Statistical analysis should include tests for normality before applying parametric tests, with appropriate corrections for multiple comparisons. For time-course studies, consider using mixed-effects models to account for both time-dependent changes and biological variability. Present data as fold-change relative to appropriate controls with error bars representing standard deviation or standard error as appropriate for the experimental design.

How can researchers resolve weak or absent signal problems when using At4g10190 antibody?

When facing weak or absent signals with At4g10190 antibody, implement a systematic troubleshooting approach. First, verify antibody viability by dot blot analysis using recombinant At4g10190 protein or positive control lysates. Next, optimize protein extraction by testing multiple buffer compositions, as At4g10190 solubility varies with subcellular localization and post-translational modifications. For Western blots, evaluate transfer efficiency using reversible staining methods such as Ponceau S. If the protein transfers poorly, adjust buffer composition (reduce methanol for larger proteins) or extend transfer time. For tissue sections or immunolocalization, implement antigen retrieval methods including citrate buffer (pH 6.0) heat treatment or Proteinase K digestion (5-10 μg/mL for 10-15 minutes). Experiment with primary antibody concentration, using a broader range than typically recommended (1:100 to 1:5000), and extend incubation times (overnight at 4°C to 48 hours for difficult antigens). Signal amplification systems such as tyramide signal amplification or polymer-based detection can increase sensitivity by 10-100 fold. For tissues with high autofluorescence, implement Sudan Black B treatment (0.1% in 70% ethanol) post-immunostaining to reduce background. Document all optimization steps systematically to identify which factors most significantly impact signal detection for At4g10190 in your specific experimental system.

What approaches can distinguish between different isoforms of At4g10190 protein in experimental samples?

Distinguishing between At4g10190 isoforms requires specialized approaches that exploit their molecular differences. First, design isoform-specific antibodies targeting unique epitopes, ideally in regions with low sequence homology. For closely related isoforms, epitope mapping using synthetic peptide arrays can identify truly isoform-specific regions. Implement high-resolution SDS-PAGE using 8-15% gradient gels to enhance separation of similarly sized isoforms. For isoforms with minimal size differences, utilize 2D gel electrophoresis to separate based on both molecular weight and isoelectric point differences. Phos-tag™ SDS-PAGE can resolve phosphorylation-dependent isoforms by selectively retarding migration of phosphorylated proteins. For immunoblotting, use highly specific monoclonal antibodies when available, or exploit differential post-translational modifications using modification-specific antibodies. Mass spectrometry offers the highest resolution for isoform identification; implement targeted proteomics approaches such as parallel reaction monitoring (PRM) to detect isoform-specific peptides with high sensitivity. When analyzing expression patterns, supplement protein data with RT-PCR using isoform-specific primers to correlate transcript and protein levels. For functional studies, develop isoform-selective knockdown strategies using RNAi targeting unique 3' UTR regions, coupled with rescue experiments using RNAi-resistant constructs expressing individual isoforms.

How can chromatin immunoprecipitation (ChIP) be optimized for At4g10190 to study DNA-binding properties?

Optimizing ChIP for At4g10190 requires tailoring standard protocols to account for plant-specific challenges and the protein's binding characteristics. Begin with appropriate crosslinking: for standard DNA-protein interactions, use 1% formaldehyde for 10 minutes at room temperature, while for proteins with weaker or indirect DNA associations, implement dual crosslinking with 1.5 mM EGS (ethylene glycol bis[succinimidylsuccinate]) for 30 minutes followed by formaldehyde. Optimize chromatin fragmentation to 200-500 bp fragments, using either sonication (for most tissues) or enzymatic digestion (for tissues resistant to sonication). For plant tissues with high polysaccharide and phenolic content, include 1% PVP-40 and 0.1% activated charcoal in the extraction buffer. Pre-clear chromatin with protein A/G beads coated with non-immune IgG to reduce background. For the immunoprecipitation step, titrate antibody concentrations between 1-10 μg per reaction to determine optimal enrichment versus background. Include appropriate controls: (1) input DNA, (2) non-immune IgG, and (3) a positive control targeting histone modifications. For qPCR analysis, design primers for both putative binding regions and negative control regions at least 5 kb away from expected binding sites. Calculate enrichment as percent input or relative to IgG control, with enrichment of >2-fold considered significant. For genome-wide analysis, implement ChIP-seq with appropriate input normalization and peak calling algorithms specific for transcription factors (MACS2) or chromatin modifiers (SICER) depending on the expected binding pattern. Validate key binding sites using electrophoretic mobility shift assays (EMSA) or reporter gene assays in planta.

How can super-resolution microscopy enhance localization studies of At4g10190?

Super-resolution microscopy techniques offer unprecedented insights into At4g10190 subcellular localization beyond the diffraction limit of conventional microscopy. Structured Illumination Microscopy (SIM) provides resolution of approximately 100 nm and is suitable for live-cell imaging of At4g10190 dynamics during pathogen response, with minimal photobleaching. For higher resolution studies, Stimulated Emission Depletion (STED) microscopy achieves 30-50 nm resolution and is particularly effective for resolving At4g10190 distribution in membrane microdomains. Single-molecule localization microscopy techniques (PALM/STORM) offer the highest resolution (10-20 nm) but require specialized fluorophores and longer acquisition times. For optimal results with At4g10190 immunolocalization, use primary antibodies at higher concentrations (1:50 dilution) than for conventional microscopy, and secondary antibodies conjugated to bright, photostable fluorophores such as Alexa Fluor 647 or Janelia Fluor dyes. Sample preparation is critical; use thin sections (≤10 μm) mounted in specialized imaging media with appropriate refractive index matching to minimize spherical aberration. For co-localization studies, implement multi-color STORM with spectral demixing algorithms to accurately distinguish between closely associated proteins. Data analysis should include cluster analysis using Ripley's K-function or DBSCAN algorithms to quantify nanoscale distribution patterns of At4g10190. Time-resolved super-resolution techniques can further elucidate the dynamic behavior of At4g10190 during signaling events, revealing transient interactions that occur below the temporal resolution of conventional microscopy.

What are the methodological considerations for using At4g10190 antibody in single-cell protein analysis techniques?

Single-cell protein analysis of At4g10190 presents unique methodological challenges that require specialized approaches. Mass cytometry (CyTOF) can be adapted for plant single-cell analysis by conjugating At4g10190 antibody to rare earth metal isotopes, typically achieving approximately 40-fold less background than fluorescence-based methods. For optimal metal labeling, use antibody concentrations of 100 μg/mL with chelating polymers such as MaxPAR or MIL. Single-cell Western blotting requires protoplast isolation using cell wall-degrading enzymes (1.5% cellulase R10, 0.5% macerozyme R10, 0.5 M mannitol, 10 mM MES pH 5.7) followed by gentle filtration through 40 μm mesh. Optimize lysis conditions (typically 0.1% SDS, 0.1% Triton X-100 in PBS) specifically for At4g10190 solubilization. For microfluidic antibody capture techniques, surface functionalization with anti-At4g10190 antibody should be performed at high density (>10,000 molecules/μm²) to overcome sensitivity limitations. Proximity ligation assay (PLA) offers enhanced sensitivity for detecting low-abundance At4g10190 in fixed single cells, with signal amplification providing up to 1000-fold enhancement over conventional immunofluorescence. For multiplexed analysis, implement cyclic immunofluorescence with antibody stripping between cycles (using 0.2 M glycine pH 2.5, 0.1% SDS, 0.1% Tween-20) to detect At4g10190 alongside other proteins of interest. Single-cell proteomics via nanoPOTS (nanodroplet Processing in One pot for Trace Samples) combined with targeted mass spectrometry can detect At4g10190 from individual plant cells with a detection limit of approximately 500 protein copies per cell. Regardless of the technique employed, extensive validation using positive and negative control cells is essential to establish specificity and sensitivity parameters for At4g10190 detection at the single-cell level.

How can researchers adapt At4g10190 antibody applications for high-throughput phenotypic screening?

Adapting At4g10190 antibody applications for high-throughput phenotypic screening requires miniaturization, automation, and quantitative analysis pipelines. Begin with automated sample preparation using robotics platforms to ensure consistent fixation, permeabilization, and antibody incubation across large sample sets. For microplate-based immunoassays, implement reverse-phase protein arrays (RPPA) in 384-well format, where cellular lysates are immobilized on nitrocellulose-coated plates and probed with At4g10190 antibody followed by near-infrared fluorescent secondary antibodies for quantitative readout. Establish Z-factor values >0.5 through optimization of positive and negative controls to ensure robust assay performance. For cell-based screening in planta, utilize automated confocal microscopy with liquid handling systems for immunofluorescence in 96-well optical-bottom plates. Optimize fixation using 4% paraformaldehyde for 15 minutes followed by 0.1% Triton X-100 permeabilization for 10 minutes, with all steps performed by liquid handling workstations. Primary antibody incubation at 1:200 dilution for 1 hour at room temperature provides adequate signal-to-noise ratio for most screening applications. Implement parallelized image analysis pipelines using CellProfiler or similar software with specific modules for nuclear segmentation, cytoplasmic identification, and quantification of At4g10190 signal intensity, localization, and pattern. For temporal dynamics, consider developing stable transgenic reporter lines expressing At4g10190 fused to HaloTag or SNAP-tag for non-antibody based visualization compatible with live-cell imaging. Multi-parametric analysis should incorporate machine learning algorithms to identify subtle phenotypic signatures associated with At4g10190 perturbation, using supervised classification models trained on positive and negative controls. Validate hits from primary screens using orthogonal assays and dose-response relationships to minimize false positives.