At1g35435 Antibody

What is At1g35435 and why are antibodies against it important in plant research?

At1g35435 is a gene locus in Arabidopsis thaliana that encodes a protein involved in plant cell signaling pathways. Antibodies targeting this protein are crucial for investigating its expression patterns, subcellular localization, and functional interactions in plant development and stress responses. Unlike general biochemical approaches, antibodies provide specificity for tracking this particular protein in complex cellular environments. These antibodies enable researchers to visualize protein distribution across tissues and quantify expression levels under various experimental conditions, offering insights into regulatory mechanisms that cannot be achieved through genetic analysis alone .

What validation protocols should be implemented before using At1g35435 antibodies in experiments?

Proper validation of At1g35435 antibodies requires a multi-step approach:

• Immunoblot analysis using both recombinant protein and plant extracts from wild-type and knockout/knockdown plants

• Peptide competition assays to confirm epitope specificity

• Cross-reactivity testing against related plant proteins

• Immunoprecipitation followed by mass spectrometry to verify target capture

For polyclonal antibodies, additional validation includes testing different bleeds and comparing reactivity patterns. A comprehensive validation should demonstrate antibody performance across multiple plant tissues and developmental stages. When using commercial antibodies, researchers should independently validate specificity rather than relying solely on manufacturer data, as antibody performance can vary across experimental conditions and sample types .

How should At1g35435 antibodies be stored to maintain optimal reactivity?

Proper storage of At1g35435 antibodies is critical for maintaining their specificity and sensitivity. For lyophilized antibodies, reconstitution should occur using sterile water or manufacturer-recommended buffers, followed by aliquoting to prevent freeze-thaw cycles. Short-term storage (<1 month) can typically be accomplished at 4°C, while long-term preservation requires -20°C or -80°C conditions . For antibodies in solution, addition of preservatives such as sodium azide (0.02%) may be necessary to prevent microbial contamination. Regular testing of antibody performance using positive controls helps monitor potential degradation over time. The following table outlines optimal storage conditions for different antibody formats:

Always spin tubes briefly before opening to collect material that may have adhered to the cap or sides .

What are optimal Western blot protocols when using At1g35435 antibodies?

For optimal Western blot results with At1g35435 antibodies, the following methodology is recommended:

Sample preparation involves extracting proteins from plant tissue using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail. Samples should be denatured at 95°C for 5 minutes in Laemmli buffer before loading onto 10-12% SDS-PAGE gels. Transfer to PVDF membranes is preferable over nitrocellulose due to stronger protein binding.

Blocking should be performed using 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature. Primary antibody incubation with At1g35435 antibody at 1:1000 dilution should occur overnight at 4°C . After washing, an appropriate secondary antibody conjugated to HRP (1:5000) is applied for 1 hour at room temperature. Signal development using enhanced chemiluminescence provides optimal sensitivity.

For challenging samples, extended blocking times (2-3 hours) and primary antibody incubation for up to 48 hours at 4°C may improve signal-to-noise ratio. Including positive and negative controls (wild-type vs. knockout plants) in each experiment is essential for result interpretation.

How can At1g35435 antibodies be effectively used in immunolocalization studies?

Immunolocalization with At1g35435 antibodies requires careful tissue preparation and antibody optimization. For plant tissues, fixation in 4% paraformaldehyde provides better epitope preservation than glutaraldehyde-based fixatives. Tissue sections should be permeabilized using a mild detergent solution (0.1% Triton X-100) to facilitate antibody access while preserving cellular structures.

Antigen retrieval methods significantly impact success rates. For At1g35435 antibodies, citrate buffer (pH 6.0) heat-mediated retrieval often yields optimal results. Primary antibody dilutions should be determined empirically, typically starting at 1:100-1:500, with overnight incubation at 4°C . Secondary antibody selection should consider fluorophore compatibility with available microscopy equipment.

To minimize background, pre-incubation of secondary antibodies with plant tissue extracts from knockout lines can absorb non-specific binding components. Counterstaining with DAPI for nuclei and specific organelle markers enables precise localization analysis. Z-stack imaging with confocal microscopy provides three-dimensional insights into protein distribution that cannot be achieved with standard fluorescence microscopy.

What controls are essential when using At1g35435 antibodies in immunoprecipitation experiments?

Immunoprecipitation (IP) experiments using At1g35435 antibodies require multiple controls to ensure data validity:

• Input control: 5-10% of pre-IP lysate should be retained to verify target protein presence

• Negative antibody control: Using non-specific IgG from the same species as the At1g35435 antibody

• Genetic control: Parallel IP from wild-type and At1g35435 knockout/knockdown plants

• Peptide competition control: Pre-incubating antibody with excess antigen peptide

• Reverse IP validation: When possible, IP with antibodies against known interaction partners

For quantitative analysis, technical replicates (minimum three) should be performed. The following workflow is recommended for optimal results:

Tissue homogenization should occur in buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, and freshly added protease inhibitors. Pre-clearing lysate with Protein A/G beads (1 hour at 4°C) reduces non-specific binding. Antibody binding should proceed overnight at 4°C with gentle rotation, followed by bead capture for 2-3 hours. Extensive washing (minimum five washes) with decreasing detergent concentrations improves specificity without losing true interactions .

How can At1g35435 antibodies be utilized in protein-protein interaction network analysis?

At1g35435 antibodies can serve as powerful tools for mapping protein interaction networks through multiple complementary approaches. For co-immunoprecipitation studies, formaldehyde crosslinking (0.5-1%) prior to cell lysis can preserve transient interactions that would otherwise be lost during purification. Sequential IPs (first with At1g35435 antibody, then with antibodies against suspected interaction partners) can confirm direct versus indirect associations.

For more comprehensive interaction mapping, antibody-based proximity labeling techniques such as BioID or APEX can be employed. In these approaches, the At1g35435 antibody is used to validate fusion protein expression and localization before proximity labeling is initiated. Mass spectrometry analysis of co-purified proteins should incorporate SAINT (Significance Analysis of INTeractome) scoring to distinguish true interactors from background contaminants .

For visualizing interactions in situ, proximity ligation assays (PLA) combining At1g35435 antibodies with antibodies against potential interaction partners can detect protein complexes with single-molecule sensitivity. The following table outlines key considerations for different protein interaction methods:

Integration of data from multiple approaches provides the most comprehensive and reliable interaction networks.

What approaches can resolve contradictory results from different At1g35435 antibody preparations?

When different At1g35435 antibody preparations yield contradictory results, a systematic troubleshooting approach is necessary:

First, comprehensively compare antibody characteristics, including host species, clonality, immunogen design, and epitope locations. Antibodies targeting different epitopes may yield different results if the protein undergoes conformational changes, post-translational modifications, or forms complexes that mask certain regions .

Perform side-by-side validation using multiple techniques. For example, if Western blot results differ between antibodies, compare their performance in immunoprecipitation, immunofluorescence, and ELISA assays. Genetic validation using CRISPR knockout lines provides the gold standard for specificity determination.

For polyclonal antibodies, affinity purification against the specific immunizing peptide can enhance specificity. If commercial antibodies are used, request technical support and lot-specific validation data from manufacturers. Additionally, independent validation by mass spectrometry can confirm the identity of bands/signals detected by different antibodies.

When discrepancies persist, report all results transparently in publications, accompanied by detailed methods and antibody information to allow proper interpretation by the scientific community.

How can At1g35435 antibodies be employed to study post-translational modifications?

When modification-specific antibodies are unavailable, a sequential immunoprecipitation strategy can be employed: first using At1g35435 antibodies to isolate the protein, followed by detection with PTM-specific antibodies (anti-phosphotyrosine, anti-ubiquitin, etc.). For phosphorylation studies, treating samples with phosphatase inhibitors during extraction is critical to preserve modification status .

Mass spectrometry analysis of immunoprecipitated At1g35435 provides comprehensive PTM mapping. This approach can identify multiple modification sites simultaneously and quantify modification stoichiometry. To enhance detection of low-abundance modified forms, enrichment steps (such as titanium dioxide for phosphopeptides) can be incorporated into the workflow.

Two-dimensional gel electrophoresis followed by Western blotting with At1g35435 antibodies can reveal charge variants resulting from phosphorylation or other modifications, providing a global view of the modified protein population without requiring modification-specific antibodies.

What are common false positive/negative results with At1g35435 antibodies and how can they be addressed?

False results with At1g35435 antibodies can arise from multiple sources and require specific troubleshooting approaches:

False Positives:

• Cross-reactivity with related proteins: Address by testing antibody reactivity in At1g35435 knockout lines and performing peptide competition assays

• Non-specific binding to other cellular components: Modify blocking conditions (try different blockers such as fish gelatin or commercial blocker formulations)

• Secondary antibody binding to endogenous plant immunoglobulins: Pre-absorb secondary antibodies with plant extract lacking primary antibody

False Negatives:

• Epitope masking by protein interactions or modifications: Try multiple antibodies targeting different epitopes

• Protein denaturation affecting antibody recognition: Test native versus denaturing conditions

• Low abundance of target protein: Implement signal amplification methods (TSA for immunofluorescence, enhanced chemiluminescence for Western blotting)

For challenging samples, protein extraction methods may need optimization, particularly for membrane-associated proteins like At1g35435. Detergent selection significantly impacts extraction efficiency, with CHAPS or digitonin sometimes preserving epitopes better than stronger detergents like SDS .

What methodological considerations are critical when using At1g35435 antibodies with different plant species or tissues?

Working with At1g35435 antibodies across different plant species or tissues requires careful methodological adaptations:

When extending from Arabidopsis to other species, sequence homology of the target protein should be evaluated, particularly in the epitope region. Even closely related species may have sequence variations that affect antibody recognition. Preliminary testing should include dot blots with recombinant proteins or tissue extracts across species of interest.

Tissue-specific considerations include cell wall composition, which affects antibody penetration for immunohistochemistry. Mature tissues may require extended permeabilization procedures or enzymatic digestion (with cellulase/pectinase) to improve accessibility. Specialized extraction buffers may be needed for recalcitrant tissues like seeds or woody stems .

Sample preparation protocols must be optimized for each tissue type:

For proteins with low abundance in specific tissues, enrichment procedures (subcellular fractionation, immunoprecipitation) may be necessary before detection with At1g35435 antibodies .

How can At1g35435 antibodies be integrated with single-cell technologies?

The integration of At1g35435 antibodies with single-cell technologies represents an emerging frontier in plant biology research. For single-cell protein analysis, multiplexed antibody-based methods such as CyTOF (mass cytometry) or MIBI (Multiplexed Ion Beam Imaging) allow simultaneous detection of At1g35435 alongside dozens of other proteins at single-cell resolution. These approaches require metal-conjugated antibodies and specialized equipment but provide unprecedented insights into cellular heterogeneity.

For combining transcriptomic and proteomic data, CITE-seq adaptations for plant systems allow simultaneous measurement of At1g35435 protein (using oligonucleotide-tagged antibodies) and mRNA in the same cells. This approach reveals potential post-transcriptional regulation by comparing protein and mRNA levels directly.

In situ approaches using highly sensitive immunofluorescence combined with RNA fluorescence in situ hybridization (FISH) can correlate At1g35435 protein localization with transcript distribution at subcellular resolution. These spatial multi-omics approaches are particularly valuable for understanding developmental processes where protein expression may be highly localized or transient .

What are the advantages and limitations of using nanobodies instead of conventional At1g35435 antibodies?

Nanobodies (single-domain antibody fragments) derived from camelid antibodies offer several advantages over conventional antibodies for At1g35435 research, but also present limitations:

Advantages:

• Smaller size (~15 kDa vs. ~150 kDa) enables better penetration into dense tissues and access to sterically hindered epitopes

• Greater stability under various pH and temperature conditions

• Simpler production in bacterial or yeast systems

• Higher-density labeling for super-resolution microscopy applications

• Less interference with protein function in live-cell applications

Limitations:

• Typically monovalent, reducing avidity compared to conventional antibodies

• Limited commercial availability specifically for plant proteins

• May recognize only a single epitope, increasing vulnerability to epitope masking

• Often require fusion tags for detection due to limited secondary antibody options

Nanobodies have been particularly successful for applications requiring minimal perturbation of protein function, such as tracking At1g35435 dynamics in living cells using fluorescently tagged nanobodies. The development of synthetic nanobody libraries shows promise for expanding available reagents for plant research .

For researchers considering nanobody development, a typical workflow involves immunizing camelids (llamas or alpacas), isolating VHH domains from peripheral blood mononuclear cells, and screening libraries through phage display or yeast two-hybrid systems .