At1g16700 Antibody

What is the At1g16700 antibody and what protein does it target?

The At1g16700 antibody is a polyclonal antibody raised in rabbits that specifically targets the At1g16700 protein from Arabidopsis thaliana (Mouse-ear cress). This protein has multiple associated names including ARABIDOPSIS THALIANA MILDEW RESISTANCE LOCUS O 14 (ATMLO14), and is also identified as NADH dehydrogenase [ubiquinone] iron-sulfur protein 8-B (mitochondrial) . The antibody recognizes specific epitopes of this protein and enables its detection in various experimental applications. The target protein has UniProt accession number Q9FX83, and understanding its role in plant biology requires specific detection tools like this antibody for investigating its expression and localization patterns .

What are the molecular characteristics of the At1g16700 antibody?

The At1g16700 antibody is a polyclonal IgG antibody produced in rabbits through immunization with recombinant Arabidopsis thaliana At1g16700 protein as the immunogen . It is supplied in liquid form with a storage buffer containing 0.03% Proclin 300 as a preservative, along with 50% glycerol and 0.01M PBS at pH 7.4 to maintain stability . The antibody undergoes antigen-affinity purification to ensure specificity while minimizing cross-reactivity with non-target proteins . This purification process is crucial for experimental reliability as it removes potential contaminants that could otherwise interfere with experimental results, particularly in sensitive applications like immunofluorescence or protein interaction studies.

What is the recommended storage protocol for maintaining At1g16700 antibody activity?

The At1g16700 antibody should be stored at either -20°C or -80°C immediately upon receipt . Repeated freeze-thaw cycles should be strictly avoided as they can significantly degrade antibody performance through protein denaturation and aggregation . For researchers planning long-term experimental series, it is advisable to create small aliquots of the antibody upon initial receipt to minimize freeze-thaw cycles. When preparing working dilutions, maintain the antibody in ice-cold conditions and use pre-chilled buffers. The storage buffer containing 50% glycerol provides cryoprotection, but proper storage temperature remains essential for preserving epitope recognition capability and preventing microbial contamination that could affect experimental outcomes.

What are the validated experimental applications for the At1g16700 antibody?

The At1g16700 antibody has been validated for enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) applications . For Western blotting, the antibody can be used to detect the native At1g16700 protein in plant tissue extracts under both reducing and non-reducing conditions. The recommended dilution for Western blotting is typically 1:1000, though optimal dilutions should be determined empirically for each experimental setup. For ELISA applications, the antibody serves as an effective detection reagent when used at appropriate dilutions determined through titration experiments. While not explicitly validated for immunofluorescence or immunohistochemistry in the provided data, researchers might consider optimization protocols for these applications based on established procedures for other plant antibodies.

What is the recommended protocol for using At1g16700 antibody in Western blotting experiments?

For Western blotting with At1g16700 antibody, begin with sample preparation by extracting total protein from Arabidopsis thaliana tissues using an appropriate extraction buffer containing protease inhibitors. Separate proteins (20-50 μg per lane) via SDS-PAGE under standard conditions. Transfer proteins to a PVDF or nitrocellulose membrane using either wet or semi-dry transfer systems. Block the membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature. Incubate with At1g16700 antibody (1:1000 dilution in blocking buffer) overnight at 4°C with gentle agitation. After washing the membrane 3-5 times with TBST, incubate with an appropriate HRP-conjugated secondary antibody (anti-rabbit IgG) at 1:5000 dilution for 1 hour at room temperature. Develop using enhanced chemiluminescence (ECL) substrate and image using a digital imaging system. The expected molecular weight of the target protein should be verified based on the protein's amino acid sequence and potential post-translational modifications.

How can researchers optimize ELISA protocols using the At1g16700 antibody?

To optimize ELISA protocols with At1g16700 antibody, researchers should first determine the optimal antibody concentration through a checkerboard titration. Begin by coating high-binding ELISA plates with plant extract containing the target protein (diluted in carbonate buffer, pH 9.6) overnight at 4°C. After washing with PBST, block plates with 3% BSA in PBS for 2 hours at room temperature. Apply the At1g16700 antibody at various dilutions (starting from 1:500 to 1:10,000) and incubate for 2 hours at room temperature. Following thorough washing, add HRP-conjugated anti-rabbit secondary antibody at a 1:5000 dilution for 1 hour. Develop with TMB substrate and stop the reaction with 2N H₂SO₄ before reading absorbance at 450 nm. Include appropriate negative controls (samples from knockout plants lacking At1g16700 expression) and positive controls (recombinant At1g16700 protein at known concentrations) to establish specificity and create standard curves for quantitative analysis.

What strategies should be employed when troubleshooting weak or non-specific signals in At1g16700 antibody applications?

When encountering weak or non-specific signals with At1g16700 antibody, several methodical troubleshooting approaches can be implemented. For weak signals, increase protein loading (50-100 μg), optimize antibody concentration (try 1:500 instead of 1:1000), extend primary antibody incubation time (overnight at 4°C), or employ signal enhancement systems like biotin-streptavidin amplification. For non-specific binding, increase blocking stringency (5-10% BSA or milk), add 0.1-0.5% Tween-20 to antibody dilution buffers, optimize washing steps (increase number and duration), or try alternative blocking agents (casein, fish gelatin). Consider using gradient gels for better protein separation and more sensitive detection methods like chemiluminescent substrates with longer exposure times. If high background persists, perform antibody purification through antigen-specific affinity columns to remove non-specific antibodies. Document all optimization steps systematically in a laboratory notebook to establish an optimized protocol for consistent results.

How can researchers quantitatively analyze Western blot data generated using the At1g16700 antibody?

For quantitative analysis of Western blot data using At1g16700 antibody, researchers should implement rigorous protocols that ensure linearity of signal response. Begin by establishing a protein loading standard curve (5-100 μg total protein) to determine the linear detection range for your specific experimental system. Include appropriate housekeeping protein controls (e.g., actin, tubulin, or GAPDH) for normalization across samples. Capture digital images of blots using a calibrated imaging system with a 16-bit dynamic range to avoid signal saturation. Perform densitometric analysis using software such as ImageJ with consistent region of interest (ROI) selections for both target and reference proteins. Calculate relative protein expression as the ratio of At1g16700 signal to housekeeping protein signal. Statistical validation should include at least three biological replicates with appropriate statistical tests (t-test or ANOVA) to determine significance of observed differences. Report results with standard deviation or standard error values and clearly indicate normalization methods in publications.

How is the At1g16700 antibody applied in studies of plant stress responses?

The At1g16700 antibody serves as a critical tool in investigating plant stress responses, particularly in relation to pathogen resistance. The target protein, associated with the MILDEW RESISTANCE LOCUS O 14, plays potential roles in plant immunity pathways . Researchers can employ the antibody to track protein expression changes under various biotic stresses, including pathogen challenges with organisms like Ralstonia solanacearum . Experimental designs typically involve exposing Arabidopsis plants to controlled stress conditions (pathogen infection, drought, temperature extremes), followed by tissue collection at defined time points post-treatment. Protein extraction and subsequent immunodetection using the At1g16700 antibody allows for temporal profiling of protein abundance changes. When combined with transcriptomic analyses, this approach enables correlation between gene expression and protein levels, providing insights into post-transcriptional regulation mechanisms during stress responses.

What considerations are important when comparing protein expression across different Arabidopsis thaliana ecotypes using the At1g16700 antibody?

When comparing protein expression across different Arabidopsis thaliana ecotypes (such as Col-5, Be-0, and Kil-0 as mentioned in the literature ), several critical considerations must be addressed. First, validate antibody cross-reactivity with the target protein across all ecotypes being studied, as natural sequence variations may affect epitope recognition. Second, implement standardized protein extraction protocols across all ecotypes, as tissue-specific composition differences might affect extraction efficiency. Third, load equal amounts of total protein (verified by Bradford or BCA assays) and confirm equal loading using multiple housekeeping proteins as references. Fourth, process all ecotype samples simultaneously on the same gel and membrane to minimize technical variation. Fifth, include appropriate internal calibration standards on each blot to enable accurate cross-blot comparisons. Finally, perform statistical analysis that accounts for both biological variation (minimum three independent biological replicates) and technical variation (minimum two technical replicates per biological sample).

How can the At1g16700 antibody be integrated into studies of protein-protein interactions in plant signaling networks?

The At1g16700 antibody can be strategically deployed in protein-protein interaction studies through several advanced methodological approaches. Co-immunoprecipitation (Co-IP) represents a primary application—researchers can use the antibody to pull down At1g16700 protein from plant extracts under native conditions, followed by mass spectrometry analysis to identify interacting protein partners. For in situ verification of interactions, proximity ligation assays (PLA) can be performed by combining the At1g16700 antibody with antibodies against suspected interaction partners. Bimolecular fluorescence complementation (BiFC) experiments can complement these approaches by expressing tagged versions of At1g16700 and potential interactors in plant protoplasts or transgenic lines. For temporal dynamics of interactions, researchers might employ fluorescence resonance energy transfer (FRET) microscopy using fluorophore-conjugated antibodies. Integration of these protein interaction data with transcriptional networks identified through methods discussed in transcriptome analysis studies can reveal comprehensive signaling pathways involved in plant defense responses, particularly in the context of MILDEW RESISTANCE functions attributed to the At1g16700 gene product.

What are the critical differences between using At1g16700 antibody in Arabidopsis research versus other plant species?

The At1g16700 antibody has been specifically validated for Arabidopsis thaliana research applications , presenting important considerations when extending its use to other plant species. Cross-reactivity with homologous proteins in other species depends primarily on epitope conservation, which requires bioinformatic analysis of protein sequence alignment and epitope prediction. Before using this antibody in non-Arabidopsis species, researchers should perform sequence homology comparisons of the At1g16700 protein across target species using tools like BLAST, focusing particularly on the immunogen sequence regions. Western blot validation using positive controls (Arabidopsis extracts) alongside the new species samples is essential to confirm cross-reactivity. If cross-reactivity exists but with lower affinity, protocol modifications including increased antibody concentration (1:500 instead of 1:1000), extended incubation periods, or more sensitive detection systems may be required. Negative results in non-Arabidopsis species should be interpreted cautiously, as they may reflect either absence of the protein or lack of antibody cross-reactivity rather than conclusive absence of homologous function.

What data management and reporting practices should researchers follow when publishing results using the At1g16700 antibody?

When publishing research utilizing the At1g16700 antibody, comprehensive reporting of methodology and validation is crucial for reproducibility. Researchers should document complete antibody information including: supplier (e.g., Cusabio), catalog number (CSB-PA887858XA01DOA), lot number, polyclonal nature, host species (rabbit), and immunogen details (recombinant Arabidopsis thaliana At1g16700 protein) . For experimental methods, specify exact antibody dilutions used (e.g., 1:1000 for Western blot), incubation conditions (time, temperature, buffer composition), detection systems, and image acquisition parameters. Include validation data demonstrating antibody specificity through controls such as knockout/knockdown lines or antigen pre-absorption tests. For quantitative analyses, detail normalization methods, statistical approaches, and replicate structure (biological vs. technical). Provide representative full-length blot images including molecular weight markers in supplementary materials, not just cropped images of bands of interest. These reporting practices align with antibody validation guidelines established by scientific journals and support the broader scientific community in reproducing and building upon the published findings.

How can At1g16700 antibody be utilized in chromatin immunoprecipitation (ChIP) experiments to study DNA-protein interactions?

While the At1g16700 antibody is primarily validated for ELISA and Western blotting , researchers interested in studying potential DNA-binding capabilities of the At1g16700 protein might adapt it for chromatin immunoprecipitation (ChIP) experiments with appropriate optimization. Begin by cross-linking DNA-protein complexes in Arabidopsis tissues using 1% formaldehyde for 10 minutes, followed by quenching with glycine. After cell lysis, sonicate chromatin to generate fragments of 200-500 bp. Pre-clear the chromatin with protein A/G beads, then incubate with At1g16700 antibody (initial test at 1:50 dilution) overnight at 4°C followed by addition of protein A/G beads. After washing, reverse cross-links and purify DNA. Validate enrichment by qPCR using primers for suspected binding regions. Optimization steps should include testing different antibody concentrations, crosslinking times, and sonication conditions. Include appropriate negative controls (IgG, non-expressing tissues) and positive controls (antibodies against known DNA-binding proteins like histones). If successful, ChIP-seq can provide genome-wide binding profiles, potentially revealing connections between At1g16700 and transcriptional regulation in mildew resistance pathways.

What methodological approaches can be used to study post-translational modifications of At1g16700 protein using the available antibody?

Investigating post-translational modifications (PTMs) of the At1g16700 protein requires specialized approaches that leverage the available antibody while accounting for potential epitope masking by modifications. First, researchers can perform immunoprecipitation using the At1g16700 antibody followed by mass spectrometry (IP-MS) to identify modifications such as phosphorylation, ubiquitination, or SUMOylation. Second, two-dimensional gel electrophoresis can separate protein isoforms based on both molecular weight and isoelectric point before Western blotting with the At1g16700 antibody, revealing charge-altering PTMs. Third, researchers can use phosphatase or deubiquitinase treatments of protein samples prior to immunodetection to confirm specific modifications through band shifts. For temporal analysis of modification events during stress responses, samples should be collected at multiple time points after treatment (e.g., pathogen infection as described in transcriptome studies ). Combining these approaches with site-directed mutagenesis of predicted modification sites in transgenic Arabidopsis lines can establish the functional significance of identified PTMs in mildew resistance and other biological processes associated with At1g16700.

How can At1g16700 antibody be incorporated into multi-omics research approaches studying plant-pathogen interactions?

Integrating the At1g16700 antibody into multi-omics frameworks provides powerful approaches for comprehensive understanding of plant-pathogen interactions, particularly relevant given the protein's association with mildew resistance . In such integrated studies, researchers can use the antibody for proteomics analysis via immunoprecipitation followed by mass spectrometry to identify protein interaction networks under normal and pathogen-challenged conditions. These proteomic datasets can be correlated with transcriptome data from RNA-seq experiments similar to those described in Arabidopsis-Ralstonia interactions , revealing post-transcriptional regulation mechanisms. Additionally, metabolomic profiles can be correlated with At1g16700 protein levels across different genetic backgrounds (wild-type vs. mutant) and treatment conditions to identify metabolic pathways influenced by the protein. For spatial context, immunolocalization using the At1g16700 antibody can determine tissue-specific expression patterns during infection. Data integration requires sophisticated computational approaches, including correlation networks, principal component analyses, and machine learning algorithms to identify key nodes in defense response networks. This systems biology approach provides holistic understanding of the protein's role within the broader context of plant immunity pathways.

Table 1: Comparison of At1g16700 Antibody Properties and Applications