At4g11393 Antibody

What is At4g11393 and why is it studied in Arabidopsis thaliana?

At4g11393 is a protein found in Arabidopsis thaliana, a small flowering plant widely used as a model organism in plant biology. While specific functional data on At4g11393 is limited in the current literature, antibodies against this protein are valuable tools for investigating its expression, localization, and interactions within plant cellular systems. The protein is referenced in the KEGG database (ath:AT4G11393), indicating its cataloging in biological pathways . Researchers typically study such proteins to understand fundamental plant biology processes, including stress responses, developmental regulation, or metabolic pathways.

What applications is the At4g11393 antibody most commonly used for?

The At4g11393 antibody is primarily used for ELISA (Enzyme-Linked Immunosorbent Assay) and Western Blot (WB) applications to detect and quantify the target protein . The antibody is antigen-affinity purified, making it suitable for specific detection of the At4g11393 protein in Arabidopsis thaliana samples. These techniques allow researchers to measure protein expression levels across different experimental conditions or to confirm protein presence in cellular fractions. The antibody's polyclonal nature makes it particularly useful for detecting native protein under various denaturing conditions that might affect epitope accessibility.

What considerations should be made when selecting fixation methods for immunohistochemistry with At4g11393 antibody?

When performing immunohistochemistry with the At4g11393 antibody, fixation protocol selection should consider preservation of both tissue morphology and epitope integrity. For plant tissues, researchers should evaluate whether aldehyde-based fixatives (paraformaldehyde or glutaraldehyde) or alcohol-based fixatives better preserve the target protein's structure. Cross-linking fixatives may mask epitopes recognized by the polyclonal At4g11393 antibody, potentially requiring antigen retrieval steps. A methodological approach would include testing different fixation times (30 minutes to overnight) and concentrations (2-4% for paraformaldehyde) to optimize the balance between structural preservation and antibody accessibility to the target protein.

How should At4g11393 antibody be stored to maintain optimal activity?

The At4g11393 antibody should be stored at -20°C or -80°C upon receipt to maintain its reactivity and specificity . Repeated freeze-thaw cycles should be avoided as these can degrade antibody quality and reduce binding efficiency. The antibody is supplied in a liquid form with a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . For working solutions, aliquoting the antibody into smaller volumes is recommended to minimize freeze-thaw cycles. When planning experiments, researchers should allow sufficient time for antibody acquisition, as this particular antibody has a lead time of 14-16 weeks since it is made-to-order .

What controls should be included when validating At4g11393 antibody specificity in Western blot applications?

When validating the At4g11393 antibody for Western blot applications, multiple controls should be implemented to ensure specificity. First, researchers should include a negative control using tissue from At4g11393 knockout mutants or tissues where the protein is not expressed. Second, a peptide competition assay where the antibody is pre-incubated with excess antigen peptide can verify binding specificity. Third, comparing the observed band size with the predicted molecular weight of At4g11393 protein provides technical validation. For more rigorous validation, researchers should consider cross-referencing results with orthogonal methods such as mass spectrometry or using multiple antibodies targeting different epitopes of the same protein. This multi-faceted approach helps distinguish between true target detection and potential cross-reactivity with similar proteins.

How can optimal antibody dilution be determined for different experimental applications?

Determining the optimal antibody dilution for the At4g11393 polyclonal antibody requires systematic titration across different experimental platforms. For Western blotting, researchers should prepare a dilution series (typically ranging from 1:500 to 1:5000) using the same protein amount and blotting conditions. For ELISA applications, a broader range of dilutions should be tested (1:100 to 1:10,000) against standardized antigen concentrations. The optimal dilution is one that produces a clear specific signal with minimal background. Signal-to-noise ratio should be calculated for each dilution by dividing the specific signal intensity by background signal intensity. Researchers should also consider that optimal dilutions may vary between different tissue types or experimental conditions due to differences in target protein abundance and sample complexity.

What sample preparation methods are recommended for detecting At4g11393 in Arabidopsis tissues?

Effective detection of At4g11393 in Arabidopsis tissues requires careful sample preparation to preserve protein integrity while removing interfering compounds. For protein extraction, researchers should use a buffer containing appropriate detergents (such as 1% Triton X-100 or 0.1-0.5% SDS), protease inhibitors, and reducing agents like DTT or β-mercaptoethanol. Plant-specific compounds like phenolics and polysaccharides can interfere with antibody binding, necessitating the addition of polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone (PVPP) to the extraction buffer. For membrane-associated proteins, more stringent extraction conditions may be required. Tissue homogenization should be performed quickly at cold temperatures to minimize protein degradation. Following extraction, protein concentration should be determined and standardized across samples to ensure comparable loading for downstream applications like Western blotting or ELISA.

How can At4g11393 antibody be utilized in co-immunoprecipitation studies to identify protein interaction partners?

Co-immunoprecipitation (Co-IP) using At4g11393 antibody requires careful optimization to preserve native protein complexes while achieving specific pulldown. Researchers should first immobilize the polyclonal At4g11393 antibody to a solid support such as protein A/G magnetic beads or agarose. The antibody concentration and coupling conditions should be optimized to ensure efficient capture without leaching during washes. Plant tissue extracts should be prepared using mild, non-denaturing lysis buffers that preserve protein-protein interactions. Pre-clearing the lysate with beads alone reduces non-specific binding. After incubating the lysate with antibody-coupled beads, a series of increasingly stringent washes removes non-specific interactions while preserving true binding partners. Eluted complexes should be analyzed by mass spectrometry for identification of interaction partners. Validation of these interactions can be performed through reciprocal Co-IP, proximity ligation assays, or fluorescence resonance energy transfer (FRET) approaches.

What strategies can overcome epitope masking when using At4g11393 antibody in fixed tissues?

Epitope masking is a common challenge when using antibodies in fixed plant tissues due to cross-linking of proteins or conformational changes. For At4g11393 detection in fixed tissues, several antigen retrieval methods can be employed. Heat-induced epitope retrieval using citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 9.0) can break protein cross-links formed during fixation. For recalcitrant samples, enzymatic retrieval using proteases like proteinase K may expose masked epitopes. The optimal method depends on the specific epitope recognized by the At4g11393 antibody and must be determined empirically. Additionally, reducing blocking buffer concentration or switching from conventional blocking agents (BSA, normal serum) to casein or commercial alternatives may improve antibody accessibility. Finally, signal amplification systems such as tyramide signal amplification can enhance detection sensitivity when epitope accessibility is limited.

How can At4g11393 antibody be employed in multiplexed immunofluorescence studies?

Multiplexed immunofluorescence with At4g11393 antibody allows simultaneous visualization of multiple proteins to study co-localization and spatial relationships. Effective multiplexing requires careful selection of compatible primary and secondary antibodies to avoid cross-reactivity. Since At4g11393 antibody is rabbit-derived , it can be paired with antibodies from different species (mouse, goat, or chicken) targeting other proteins of interest. Secondary antibodies with minimal cross-reactivity and spectrally distinct fluorophores should be selected. Sequential staining protocols can be implemented when using multiple rabbit-derived antibodies, employing complete elution of the first set of antibodies before applying the second set. Advanced detection systems like tyramide signal amplification allow same-species antibodies to be used sequentially. Multi-round imaging with antibody stripping and reprobing can further expand multiplexing capabilities, although careful validation is needed to ensure complete antibody removal between rounds.

What are potential causes and solutions for high background when using At4g11393 antibody in immunohistochemistry?

High background in immunohistochemistry with At4g11393 antibody can stem from multiple factors. Insufficient blocking is a common cause, requiring extended blocking times (2+ hours) or higher concentrations of blocking agents (5% BSA or 10% normal serum). The presence of endogenous peroxidase activity in plant tissues can be addressed by including a hydrogen peroxide treatment step (3% H₂O₂ for 10-15 minutes) before antibody incubation. Non-specific binding due to hydrophobic interactions can be reduced by adding 0.1-0.3% Triton X-100 or Tween-20 to washing and incubation buffers. Excessive antibody concentration often contributes to background; titrating to lower concentrations while extending incubation time (overnight at 4°C) can improve signal-to-noise ratio. Autofluorescence from plant cell walls and chlorophyll can be minimized using specific quenching agents like Sudan Black B (0.1-0.3%) or sodium borohydride treatment. Additionally, adjusting the detection system from chromogenic to fluorescent methods or vice versa may improve signal discrimination.

How should researchers address inconsistent Western blot results with At4g11393 antibody?

Inconsistent Western blot results with At4g11393 antibody may derive from several methodological variables. Sample preparation inconsistencies can be addressed by standardizing protein extraction protocols, including rapid harvesting on ice, consistent buffer-to-tissue ratios, and verified protein quantification methods. Transfer efficiency variation can be minimized by using stain-free technology or Ponceau S staining to confirm uniform protein transfer. For the antibody itself, creating single-use aliquots prevents degradation from freeze-thaw cycles, while lot-to-lot variations can be tracked through detailed record-keeping. The polyclonal nature of the At4g11393 antibody may introduce batch variations; maintaining a reference sample across experiments allows for normalization. Protein modifications affecting epitope recognition can be investigated using phosphatase treatment (for phosphorylation) or deglycosylation enzymes (for glycosylation). Finally, optimization of incubation conditions through temperature adjustment (4°C overnight versus room temperature for 1-2 hours) and buffer composition modifications (varying salt concentration or detergent type) can significantly improve consistency.

What modifications to protocols are needed when using At4g11393 antibody across different Arabidopsis ecotypes or mutant lines?

When applying At4g11393 antibody across different Arabidopsis ecotypes or mutant lines, protocol modifications may be necessary to accommodate genetic and physiological variations. Protein expression levels can vary significantly between ecotypes, requiring adjustment of antibody concentration or exposure times. Extraction buffer composition should be optimized for each ecotype, potentially requiring higher detergent concentrations for lines with different cell wall compositions. Differences in post-translational modifications between ecotypes might affect epitope accessibility, necessitating modified antigen retrieval methods. When working with transgenic lines expressing tagged versions of At4g11393, researchers should verify that the tag doesn't interfere with antibody binding. For quantitative comparisons between ecotypes, normalization to multiple housekeeping proteins is essential to account for loading variations. Additionally, researchers should sequence the At4g11393 gene across ecotypes to identify potential amino acid changes that might affect antibody recognition, particularly if unexpected results are observed.

How can At4g11393 antibody be effectively utilized in chromatin immunoprecipitation (ChIP) experiments?

Utilizing At4g11393 antibody in chromatin immunoprecipitation requires specialized protocol adaptations for plant chromatin. Researchers should begin with optimized cross-linking conditions specifically for Arabidopsis tissues, typically using 1-2% formaldehyde for 10-15 minutes. The sonication parameters must be carefully calibrated to achieve chromatin fragments of 200-500 bp while maintaining protein epitope integrity. Pre-clearing with protein A beads (given the rabbit origin of the antibody ) reduces non-specific binding. Researchers should determine the optimal antibody-to-chromatin ratio through titration experiments, typically starting with 2-5 μg antibody per immunoprecipitation. Stringent washing steps with increasing salt concentrations help eliminate false positives. ChIP-qPCR validation of enriched regions should precede genome-wide sequencing approaches. As At4g11393 is likely not a transcription factor itself, this approach would be valuable for exploring its potential association with chromatin, possibly through interaction with DNA-binding proteins or as part of chromatin-modifying complexes. Controls should include IgG pulldowns and input samples, as well as positive controls targeting known chromatin-associated proteins.

What considerations are important when designing super-resolution microscopy experiments with At4g11393 antibody?

Super-resolution microscopy with At4g11393 antibody demands careful attention to sample preparation and fluorophore selection. Plant cell walls and vacuoles present unique challenges for techniques like STORM, PALM, or STED microscopy. Sample preparation should minimize autofluorescence through treatments like 0.1% sodium borohydride or specific quenching agents. The secondary antibody should be conjugated to bright, photostable fluorophores compatible with the chosen super-resolution technique (e.g., Alexa Fluor 647 for STORM or ATTO 647N for STED). Mounting media composition critically affects fluorophore blinking kinetics in STORM/PALM approaches and should contain appropriate oxygen scavenging systems and thiol compounds. Drift correction and multichannel alignment require fiducial markers integrated into the sample. For quantitative analysis, clustering algorithms should be selected based on the expected biological distribution of At4g11393. Control experiments should include secondary-only samples and known structures of similar scale to validate resolution claims. Importantly, researchers should verify that the secondary detection system doesn't limit resolution beyond the primary antibody size (approximately 10-15 nm), which often becomes the limiting factor in antibody-based super-resolution studies.

How can At4g11393 antibody be adapted for proximity-dependent labeling approaches like BioID or APEX?

Adapting At4g11393 antibody for proximity-dependent labeling requires strategic fusion construct design and validation. Rather than directly using the antibody, researchers would generate fusion constructs between At4g11393 and enzymatic tags like BioID2 (a biotin ligase) or APEX2 (an ascorbate peroxidase). These constructs should be validated for proper expression, localization, and functionality using the At4g11393 antibody before proximity labeling experiments. The antibody serves as a crucial validation tool to confirm that the fusion protein maintains native localization patterns and expression levels comparable to endogenous At4g11393. For temporal control, inducible expression systems can be employed. Following proximity labeling and affinity purification of biotinylated or tagged proteins, mass spectrometry identifies potential interaction partners. Validation of these interactions should include co-localization studies using the At4g11393 antibody alongside antibodies against identified partners. Careful control experiments, including using untargeted BioID/APEX enzymes localized to the same subcellular compartment, help distinguish true proximity interactions from background labeling.