At4g11655 Antibody

What is At4g11655 and why is it significant in plant research?

At4g11655 refers to a specific gene locus in the Arabidopsis thaliana genome, located on chromosome 4. This gene encodes a protein that plays significant roles in plant cellular processes. Antibodies targeting this protein are valuable tools for researchers investigating plant cellular signaling, protein localization, and molecular interactions. The significance of At4g11655 stems from its involvement in environmental response pathways, similar to other PAS domain-containing proteins that sense environmental stimuli such as oxygen, redox conditions, or light . Research using At4g11655 antibodies allows visualization and quantification of this protein in various experimental contexts, enabling deeper understanding of plant adaptation mechanisms and stress responses.

What sample preparation protocols optimize At4g11655 antibody performance in Western blotting?

For optimal Western blotting results with At4g11655 antibody, sample preparation should focus on preserving protein integrity while maximizing extraction efficiency. Begin with flash-freezing plant tissue in liquid nitrogen followed by grinding to fine powder. Extract proteins using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and freshly added protease inhibitors. For phosphorylation studies, include phosphatase inhibitors (10 mM NaF, 1 mM Na3VO4). Centrifuge at 14,000×g for 15 minutes at 4°C and collect the supernatant. Quantify protein concentration using Bradford or BCA assay before loading 10-30 μg per lane. Use non-reducing conditions when possible, as reducing agents may affect epitope recognition . For membrane-associated proteins, consider specialized extraction buffers containing 0.1% SDS to improve solubilization without denaturing the target epitope.

How should At4g11655 antibody be stored and handled to maintain reactivity?

Proper storage and handling of At4g11655 antibody is crucial for maintaining its reactivity and specificity. Upon receipt, store lyophilized antibody immediately at -20°C in a manual defrost freezer . After reconstitution with 0.1 ml distilled water, continue storage at -20°C in small aliquots to minimize freeze-thaw cycles . For long-term storage beyond 6 months, adding 0.09% sodium azide as a preservative is recommended, though this should be avoided for functional assays due to potential interference . Avoid storage in frost-free freezers where temperature fluctuations can gradually denature the antibody . Prior to use, centrifuge any solutions containing precipitates. Working dilutions should be prepared fresh and used within 24 hours. For shipping or temporary storage, 4°C is acceptable for up to one week, but longer periods require freezing to preserve antibody activity.

What controls are essential when validating At4g11655 antibody specificity?

Rigorous validation of At4g11655 antibody specificity requires multiple complementary controls. First, include a positive control using recombinant At4g11655 protein alongside your experimental samples. Second, implement a pre-absorption control by pre-incubating the antibody with excess target antigen before immunostaining, which should abolish specific signals. Third, include samples from knockout/knockdown mutant lines lacking At4g11655 expression, which should show significantly reduced or absent signal. Fourth, perform parallel experiments with an alternate antibody targeting a different epitope of the same protein to confirm staining patterns. Fifth, run a Western blot to verify that the antibody detects a band of the expected molecular weight. Technical controls should include a secondary-antibody-only control to assess non-specific binding and samples from phylogenetically related species to evaluate cross-reactivity. These comprehensive controls collectively establish antibody specificity and reliability across different experimental contexts.

How can researchers optimize immunohistochemistry protocols for At4g11655 detection in plant tissues?

Successful immunohistochemistry for At4g11655 detection in plant tissues requires careful optimization. Begin with thorough fixation using 4% paraformaldehyde in PBS for 12-24 hours, followed by paraffin embedding. Cut sections at 5-7 μm thickness and mount on positively charged slides. Critical for plant tissues is proper antigen retrieval—heat-mediated treatment in citrate buffer (pH 6.0) is recommended . Block with 5% normal serum matching the secondary antibody host species plus 0.3% Triton X-100 in PBS for 1 hour. Apply primary At4g11655 antibody at 5.0 μg/ml concentration in blocking solution and incubate overnight at 4°C . After thorough washing (3 × 10 minutes in PBS), apply appropriate biotinylated secondary antibody, followed by streptavidin-HRP complex and chromogenic substrate. For fluorescent detection, use fluorophore-conjugated secondary antibodies and include DAPI nuclear counterstain. Plant-specific considerations include minimizing autofluorescence by treating sections with 0.1% sodium borohydride or 0.3% Sudan Black B in 70% ethanol before antibody application. Process control and experimental tissues identically for valid comparisons.

What dilution optimization strategies ensure reproducible ELISA results with At4g11655 antibody?

Achieving reproducible ELISA results with At4g11655 antibody requires systematic dilution optimization. Begin with a checkerboard titration using a dilution series of both capture antibody (0.5-5 μg/ml) and detection antibody (0.1-2 μg/ml) . Perform this optimization with known quantities of recombinant At4g11655 protein ranging from 0-1000 pg/ml to establish standard curves at each dilution combination. Select optimal dilutions based on maximum signal-to-noise ratio and linear dynamic range rather than maximum signal intensity alone. For sandwich ELISA specifically, use purified At4g11655 antibody as the capture reagent, and biotinylated At4g11655 antibody as the detection reagent . Include internal standards on every plate to normalize plate-to-plate variation. Once optimal dilutions are established, validate by testing intra-assay precision (CV < 10% for replicate wells) and inter-assay precision (CV < 15% across plates). Document the detailed dilution protocol, including buffer compositions, incubation times, and temperatures to ensure consistent reproduction of results across experiments.

How can researchers address cross-reactivity issues with At4g11655 antibody?

Cross-reactivity represents a significant challenge when working with At4g11655 antibody, particularly given the homology between proteins containing similar domains. To address this issue, first characterize the extent of cross-reactivity by testing the antibody against recombinant proteins with similar domains or close homologs. If cross-reactivity is detected, implement several strategies: First, try antibody pre-adsorption with the cross-reactive protein to deplete non-specific binding, carefully titrating the amount used to maintain specific binding while eliminating cross-reactivity. Second, optimize blocking conditions using 5% BSA rather than serum-based blockers, which may introduce additional cross-reactive epitopes. Third, increase stringency in immunoprecipitation experiments by using higher salt concentrations (up to 500 mM NaCl) in wash buffers. Fourth, for Western blotting, consider using gradient gels that provide better separation of similarly sized proteins. Fifth, validate results using orthogonal methods such as mass spectrometry to confirm protein identity. Most importantly, acknowledge any remaining cross-reactivity in research publications, precisely documenting the extent and limitations of antibody specificity.

What strategies can overcome low signal strength in At4g11655 antibody applications?

Low signal strength with At4g11655 antibody can stem from multiple factors requiring systematic troubleshooting. First, evaluate antibody viability by testing with positive control recombinant protein. If the problem persists, implement signal amplification strategies: For Western blotting, switch to high-sensitivity chemiluminescent substrates or consider using femto-level detection reagents that can increase sensitivity by 10-50 fold. For immunohistochemistry, implement tyramide signal amplification (TSA), which can enhance sensitivity up to 100-fold through catalyzed reporter deposition. Optimize antigen retrieval methods systematically, testing both heat-mediated approaches with citrate buffer (pH 6.0) and enzymatic methods using proteinase K . For particularly challenging applications, increase primary antibody concentration incrementally while monitoring background levels. Extend primary antibody incubation time to overnight at 4°C to enhance binding without increasing background. For extraction from recalcitrant plant tissues, incorporate specialized buffers containing 0.5-1% SDS or 8M urea for improved protein solubilization and epitope exposure. Finally, evaluate whether target protein abundance varies with developmental stage or environmental conditions, and adjust experimental design accordingly.

How do freeze-thaw cycles affect At4g11655 antibody performance, and what mitigation strategies exist?

Repeated freeze-thaw cycles significantly impact At4g11655 antibody performance through progressive degradation of protein structure. Each cycle can reduce antibody activity by 5-20%, with cumulative effects leading to complete loss of functionality. When subjected to multiple freeze-thaw cycles, antibodies undergo partial denaturation, aggregate formation, and fragmentation of heavy and light chains, all contributing to decreased specificity and sensitivity. To mitigate these effects, implement several protective measures: First, aliquot newly reconstituted antibody into single-use volumes (typically 10-20 μl) in siliconized tubes to prevent protein adsorption to vessel walls . Second, use cryoprotectants such as 50% glycerol when preparing working dilutions for storage. Third, thaw aliquots rapidly at room temperature rather than at 37°C to minimize time spent at intermediate temperatures where degradation occurs most rapidly. Fourth, add carrier proteins like 0.1-1% BSA to dilute antibody preparations to prevent adsorptive losses. Fifth, consider storage in antibody stabilizing solutions containing non-toxic preservatives when preparing working dilutions. Document the number of freeze-thaw cycles for each aliquot and establish maximum thresholds through empirical testing of antibody performance after various numbers of cycles.

How can At4g11655 antibody be effectively used in co-immunoprecipitation to identify interaction partners?

Co-immunoprecipitation (Co-IP) with At4g11655 antibody requires careful optimization to maintain native protein interactions while achieving sufficient specificity. Begin with gentle lysis conditions using buffers containing 0.5% NP-40 or 1% digitonin rather than stronger detergents that may disrupt protein-protein interactions. Pre-clear lysates with protein A/G beads to reduce non-specific binding. For the immunoprecipitation, covalently cross-link the At4g11655 antibody to protein A/G beads using dimethyl pimelimidate to prevent antibody heavy and light chains from appearing in the final analysis, which could mask interacting proteins of similar molecular weights. Incubate the cross-linked antibody-bead complex with pre-cleared lysate overnight at 4°C with gentle rotation. After stringent washing, elute protein complexes with non-reducing elution buffer to preserve complex integrity. For identification of novel interaction partners, submit samples for mass spectrometry analysis, comparing with appropriate controls including immunoprecipitation with non-specific IgG and samples from At4g11655 knockout plants. Validate identified interactions through reciprocal Co-IP experiments using antibodies against the putative interacting partners, followed by confirmatory approaches such as bimolecular fluorescence complementation or FRET assays.

What considerations are important when using At4g11655 antibody for chromatin immunoprecipitation (ChIP) experiments?

Chromatin immunoprecipitation (ChIP) with At4g11655 antibody presents unique challenges requiring specialized optimization. First, ensure the antibody recognizes its epitope in a fixed chromatin context by performing pilot experiments with increasing formaldehyde concentrations (0.5-3%) and fixation times (5-20 minutes). For plant tissues specifically, include a vacuum infiltration step during fixation to ensure penetration through waxy cuticles. Sonication parameters require careful optimization—aim for chromatin fragments between 200-500 bp, checking fragment size distribution by agarose gel electrophoresis. Use 5-10 μg of At4g11655 antibody per ChIP reaction, substantially more than typical for Western blotting. Critical controls include input chromatin (pre-immunoprecipitation), non-specific IgG precipitation, and samples from At4g11655 knockout plants. For qPCR analysis of precipitated DNA, design primers for both putative binding regions and negative control regions (typically gene deserts). Calculate enrichment as percent input normalized to IgG control values. For ChIP-seq applications, perform IP in at least biological triplicates and prepare libraries from 5-10 ng of precipitated DNA. During bioinformatic analysis, use appropriate peak-calling algorithms suitable for transcription factor binding (e.g., MACS2) with stringent parameters (q-value < 0.01) to minimize false positives.

How can At4g11655 antibody be validated for super-resolution microscopy applications?

Validating At4g11655 antibody for super-resolution microscopy requires additional specificity and performance criteria beyond conventional imaging approaches. Begin with fixation method optimization, testing both chemical (paraformaldehyde, glutaraldehyde) and cryo-fixation techniques to determine which best preserves native protein localization while maintaining epitope accessibility. For STORM or PALM microscopy, evaluate antibody performance with direct fluorophore conjugation versus secondary antibody detection, as the physical distance between protein and fluorophore significantly impacts achievable resolution. Test antibody specificity under super-resolution conditions by performing parallel experiments in wild-type and knockout/knockdown plant tissues, analyzing not just presence/absence of signal but also comparing spatial distribution patterns at nanometer scale. Quantify labeling density (signals per μm²) and estimate localization precision through technical replicates. For dual-color applications, perform rigorous cross-talk controls and measure chromatic aberration using fiducial markers. Validate biological findings by correlating super-resolution data with orthogonal approaches such as electron microscopy with immunogold labeling or biochemical fractionation. Document detailed protocols including sample preparation, imaging parameters, and analysis algorithms to ensure reproducibility, as super-resolution techniques are particularly sensitive to minor procedural variations.

How does polyclonal versus monoclonal At4g11655 antibody selection impact experimental outcomes?

The choice between polyclonal and monoclonal At4g11655 antibodies significantly impacts experimental outcomes across various applications. Polyclonal antibodies recognize multiple epitopes on the At4g11655 protein, providing higher sensitivity through signal amplification but potentially increased background and batch-to-batch variation. Monoclonal antibodies target a single epitope, offering greater specificity but potentially reduced sensitivity, especially if the epitope is masked in certain experimental conditions.

For applications requiring highest specificity (e.g., distinguishing between closely related protein family members), monoclonal antibodies are preferable. For detection of low-abundance proteins or applications involving partial protein denaturation, polyclonal antibodies often provide superior results. Ideally, researchers should validate both types in preliminary experiments before selecting the optimal antibody for their specific research application.

What epitope considerations influence At4g11655 antibody selection for different applications?

Epitope characteristics critically influence At4g11655 antibody performance across different applications, requiring application-specific selection criteria. For Western blotting applications, antibodies recognizing linear epitopes perform optimally, as proteins are denatured during SDS-PAGE . Conversely, for applications using native proteins (immunoprecipitation, ChIP, flow cytometry), antibodies recognizing conformational epitopes better maintain specificity. When targeting post-translational modifications (PTMs), epitope location relative to the modification site is crucial—antibodies with epitopes too close to PTM sites may show differential binding to modified versus unmodified protein.

For plant proteins specifically, consider epitopes that avoid regions with high homology to related family members. Additionally, epitopes in regions prone to proteolytic cleavage during sample preparation should be avoided, particularly when working with protease-rich plant tissues. Custom epitope design targeting unique regions of At4g11655 may be necessary when commercial options show insufficient specificity.

How do different secondary antibody conjugates affect detection sensitivity in At4g11655 immunoassays?

Secondary antibody conjugate selection substantially impacts detection sensitivity and signal-to-noise ratio in At4g11655 immunoassays. The choice of conjugate should align with detection method, required sensitivity, and potential background interference from the specific plant tissue being analyzed.

For plant tissues specifically, conjugates enabling signal amplification (such as HRP with tyramide signal amplification or biotin with streptavidin systems) often overcome challenges related to low target abundance . When working with green tissues, avoid fluorophores with emission spectra overlapping chlorophyll autofluorescence (primarily 650-700 nm range). For multiplex detection, carefully select fluorophores with minimal spectral overlap or consider sequential detection with HRP conjugates and different chromogenic substrates.

How can At4g11655 antibody be effectively utilized in plant stress response studies?

At4g11655 antibody offers valuable insights into plant stress response mechanisms through multiple methodological approaches. For drought stress studies, implement time-course experiments comparing At4g11655 protein levels in drought-stressed versus well-watered plants using quantitative Western blotting with loading controls such as anti-actin or anti-tubulin antibodies. Complement protein expression data with subcellular localization studies using immunofluorescence microscopy to track potential stress-induced relocalization of At4g11655. Given that many PAS domain-containing proteins sense environmental stimuli such as oxygen and redox conditions , At4g11655 may show altered interaction patterns under stress conditions, making co-immunoprecipitation followed by mass spectrometry particularly informative. For field-relevant stress conditions, consider combining controlled environment studies with field sampling across natural stress gradients, preserving protein integrity through immediate flash-freezing of collected tissues. Experimental design should include both acute and chronic stress treatments to distinguish between immediate signaling responses and adaptive changes. Correlate protein-level changes with transcriptomic data to identify potential post-transcriptional regulation mechanisms. These approaches collectively provide mechanistic insights into At4g11655's role in plant environmental adaptation.

What methodological approaches enable quantitative analysis of At4g11655 protein levels across developmental stages?

Quantitative analysis of At4g11655 protein across developmental stages requires rigorous methodological approaches to ensure accurate comparison. Implement a systematic tissue sampling strategy covering key developmental stages from germination through flowering and senescence with consistent harvesting protocols including time of day and plant position standardization. For protein extraction, use a single unified protocol across all developmental stages, with buffer modifications as necessary for tissues with varying compositions (higher detergent concentrations for waxy tissues, additional protease inhibitors for proteolytically active tissues). Quantitative Western blotting represents the foundation of analysis, using infrared fluorescence detection systems (e.g., LI-COR) that provide broader linear dynamic range than chemiluminescence. Critical for developmental comparisons is proper normalization, ideally using multiple reference proteins validated for stable expression across developmental stages.

Complement Western blot data with quantitative immunohistochemistry to reveal tissue-specific expression patterns, calculating signal intensity normalized to tissue area across developmental stages. For highest precision, consider parallel reaction monitoring mass spectrometry using isotope-labeled peptide standards matching At4g11655 tryptic fragments.

How can At4g11655 antibody be integrated into high-throughput phenotypic screening platforms?

Integrating At4g11655 antibody into high-throughput phenotypic screening requires streamlined workflows balancing throughput with data quality. Develop an automated tissue sampling system using plant growth in standard multi-well formats with robotic harvesting capabilities. For protein extraction, implement a magnetic bead-based protocol compatible with liquid handling systems, using paramagnetic beads conjugated to At4g11655 antibody for direct immunoprecipitation from crude lysates. Convert traditional Western blotting to capillary-based automated systems (e.g., ProteinSimple Wes) that require minimal sample volume (3 μl) while providing quantitative results for hundreds of samples daily.

For cellular phenotyping, establish high-content imaging workflows using At4g11655 antibody in combination with other phenotypic markers in a multiplexed immunofluorescence format. Implement machine learning algorithms for automated image analysis, trained to recognize subcellular distribution patterns of At4g11655 across treatment conditions. For genetic screening applications, create a reporter system where At4g11655 antibody is used to validate hits from primary screens through automated ELISA performed in 384-well format.

Design custom microfluidic devices for super-high-throughput applications, enabling single-cell protein quantification through antibody-based detection combined with microfluidic sorting. These technologies collectively transform traditional low-throughput biochemical analysis into scalable screening platforms capable of processing thousands of conditions while maintaining the specificity of antibody-based detection, facilitating systems-level understanding of At4g11655 function across genetic backgrounds and environmental conditions.