At1g65740 Antibody

What is the At1g65740 gene and its encoded protein in Arabidopsis thaliana?

At1g65740 is a gene locus in Arabidopsis thaliana that encodes a specific protein. While the exact protein isn't directly mentioned in the provided search results, we can understand the context of plant protein studies using similar approaches. In Arabidopsis research, proteins are typically characterized by their gene loci (such as At1g65740), molecular weight, and function. Similar to other characterized Arabidopsis proteins, it would be studied using recombinant protein expression and antibody-based detection methods . The characterization typically includes protein length (in amino acids), molecular weight (kDa), and predicted biological function based on sequence homology or experimental evidence.

How are antibodies against Arabidopsis proteins typically generated?

Antibodies against Arabidopsis proteins are typically generated by first cloning the target cDNA into an expression vector, such as a GATEWAY-compatible Escherichia coli expression vector . The recombinant proteins are expressed with tags (commonly RGS-His6 tags) to facilitate purification. After protein expression induction with IPTG and harvesting by centrifugation, the purified proteins are used as antigens for antibody production . Both monoclonal and polyclonal antibodies can be generated depending on the research needs. For monoclonal antibodies, hybridoma technology is employed, while polyclonal antibodies are typically raised in rabbits, rats, or mice by immunization with the purified protein antigens.

What are the primary applications of plant protein antibodies in Arabidopsis research?

Plant protein antibodies in Arabidopsis research serve multiple critical functions. First, they enable protein detection in Western blots, immunoprecipitation, and immunolocalization studies to track expression patterns across tissues and developmental stages. Second, antibodies are essential tools for investigating protein-protein interactions through co-immunoprecipitation experiments . Third, they allow for functional characterization of proteins in the context of plant immune responses, as seen in studies of effector-triggered immunity in Arabidopsis . Fourth, antibodies are valuable for validating protein expression from transgenic constructs. Finally, using protein chips and antibodies, researchers can perform high-throughput screening of thousands of Arabidopsis proteins simultaneously, drastically accelerating functional genomics research .

How can I assess and mitigate antibody cross-reactivity with other Arabidopsis proteins?

Cross-reactivity assessment is crucial for ensuring antibody specificity, particularly when working with protein families that share sequence similarity. To evaluate cross-reactivity, utilize Arabidopsis protein chips containing multiple family members, as demonstrated with MYB and DOF transcription factor families . These chips allow screening against numerous proteins simultaneously to identify potential cross-reactions. Additionally, perform Western blots using extracts from knockout/knockdown lines of your target gene as negative controls. For mitigation strategies, consider antibody purification through affinity chromatography using immobilized antigen. Alternatively, epitope-specific antibodies targeting unique regions of the protein can be developed. When designing immunization strategies, select protein fragments excluding conserved domains to reduce cross-reactivity probability. Finally, validation through multiple detection methods (Western blotting, immunoprecipitation, and immunohistochemistry) strengthens confidence in antibody specificity.

What are the optimal conditions for immunoprecipitation using At1g65740 antibody?

Optimizing immunoprecipitation (IP) conditions requires systematic testing of multiple parameters. Based on protocols used for similar Arabidopsis proteins, start with tissue grinding in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, and protease inhibitor cocktail . Pre-clear lysates by centrifugation at 14,000 × g for 15 minutes at 4°C. For antibody binding, incubate cleared lysates with antibody (2-5 μg) overnight at 4°C with gentle rotation. Capture complexes using protein A/G magnetic beads for 2 hours at 4°C. Perform four sequential washes with decreasing salt concentrations (from 300 mM to 150 mM NaCl). Elute proteins by boiling in SDS sample buffer or using acidic glycine buffer (pH 2.8) for native elution. Always include IgG controls from the same species to identify non-specific binding. For weak interactions, consider crosslinking approaches using formaldehyde or DSP (dithiobis(succinimidyl propionate)).

How can At1g65740 antibody be used to investigate plant immune responses?

At1g65740 antibody can be instrumental in investigating plant immune responses through multiple approaches. First, use immunoblotting to track protein abundance changes during immune activation, such as after pathogen infection or PAMP (Pathogen-Associated Molecular Pattern) treatment. Studies of effector-triggered immunity in Arabidopsis show dramatic changes in host hormone signaling and altered expression of thousands of genes , which likely affects protein levels. Second, employ immunoprecipitation followed by mass spectrometry (IP-MS) to identify immune-related interaction partners that associate with the protein during defense responses. Third, perform chromatin immunoprecipitation (ChIP) if the protein has DNA-binding capabilities to identify genomic targets during immune activation. Fourth, use immunohistochemistry to visualize protein localization changes during pathogen challenge, potentially revealing translocation events important for defense signaling. Finally, combine antibody-based approaches with genetic studies using knockouts and overexpression lines to establish functional relationships in immune signaling networks.

What are the recommended protocols for Western blot detection using At1g65740 antibody?

For optimal Western blot detection using At1g65740 antibody, follow this comprehensive protocol based on successful approaches with Arabidopsis proteins . First, extract total protein from 100 mg plant tissue using 300 μl extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM PMSF, and protease inhibitor cocktail). Determine protein concentration using Bradford assay. Load 20-30 μg protein per lane on a 10-12% SDS-PAGE gel and separate at 120V. Transfer proteins to PVDF membrane (100V for 1 hour) in Towbin buffer with 20% methanol. Block membrane with 5% non-fat dry milk or 2% BSA in TBST for 1 hour at room temperature . Incubate with primary antibody (1:1000-1:2000 dilution) overnight at 4°C. Wash four times with TBST, 10 minutes each. Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature. Wash thoroughly and develop using ECL substrate. For troubleshooting, include positive controls (recombinant protein) and test different extraction buffers if initial detection is weak.

How can I optimize immunohistochemistry protocols for plant tissues using At1g65740 antibody?

Optimizing immunohistochemistry for plant tissues requires addressing the unique challenges of plant cell walls and autofluorescence. Begin with tissue fixation in 4% paraformaldehyde in PBS for 2 hours at room temperature or overnight at 4°C. For better penetration, include 0.1% Triton X-100 in the fixative. After fixation, wash tissues three times in PBS and proceed with embedding in paraffin or freeze in OCT compound for cryosectioning. Cut sections at 8-12 μm thickness. For paraffin sections, perform antigen retrieval by heating sections in citrate buffer (pH 6.0) for 10 minutes at 95°C to unmask epitopes. Block non-specific binding with 5% normal serum and 2% BSA in PBS with 0.1% Triton X-100 for 1 hour. Apply primary antibody (1:100-1:500 dilution) and incubate overnight at 4°C in a humid chamber. After washing, apply fluorophore-conjugated secondary antibody (1:200-1:500) for 2 hours at room temperature. To reduce plant autofluorescence, treat sections with 0.1% Sudan Black B in 70% ethanol for 10 minutes prior to mounting. Include controls with pre-immune serum and secondary antibody alone to assess background.

What approaches can be used to validate the specificity of At1g65740 antibody?

Validating antibody specificity requires multiple complementary approaches. First, perform Western blot analysis comparing wild-type plants with knockout/knockdown mutants of At1g65740; specific antibodies should show reduced or absent signal in mutant lines . Second, conduct peptide competition assays by pre-incubating the antibody with excess purified antigen before immunodetection; specific binding should be blocked by this treatment. Third, compare detection patterns with antibodies raised against different epitopes of the same protein; consistent patterns indicate specificity. Fourth, use protein arrays containing multiple Arabidopsis proteins to assess cross-reactivity, as demonstrated with anti-TCP1, anti-MYB6, and anti-DOF11 antibodies . Fifth, validate through immunoprecipitation followed by mass spectrometry to confirm the identity of the pulled-down protein. Finally, utilize heterologous expression systems to express tagged versions of the protein and confirm co-detection with both anti-tag and anti-protein antibodies. Document all validation steps thoroughly, including experimental conditions and controls, to establish confidence in antibody performance.

How can At1g65740 antibody be integrated with protein microarray technologies?

Integration of At1g65740 antibody with protein microarray technologies enables high-throughput screening of protein-protein interactions and post-translational modifications. Based on established Arabidopsis protein chip methodologies , purify the antibody to high specificity using affinity chromatography. For direct antibody printing, use robotic arrayers to spot the purified antibody onto nitrocellulose-coated FAST slides or polyacrylamide-coated PAA slides at concentrations of 0.1-1 mg/ml . Alternatively, for reverse-phase arrays, print plant protein extracts and probe with the antibody. The detection limit on these platforms is approximately 2-3.6 fmol per spot on FAST slides or 0.1-1.8 fmol per spot on PAA slides . For fluorescence detection, use secondary antibodies labeled with Cy3 or Cy5 fluorophores at 1:800 dilution . Always include technical controls such as spotted IgG and secondary antibody-only controls. For quantitative analysis, implement standard curves using purified recombinant protein. This approach enables simultaneous analysis of protein abundance across multiple experimental conditions or genetic backgrounds.

What are the best preservation methods for long-term storage of At1g65740 antibody?

For optimal long-term preservation of antibody functionality, implement a multi-tier storage strategy. Primary storage should be in small aliquots (20-50 μl) at -80°C in PBS or TBS buffer with 50% glycerol and 0.02% sodium azide as a preservative. This prevents repeated freeze-thaw cycles that can denature antibodies. For working stocks, maintain aliquots at -20°C with 50% glycerol. If frequent use is anticipated, keep a small working aliquot at 4°C for up to one month, but monitor for signs of decreased activity. For lyophilization, freeze the antibody solution with 1-2% trehalose or sucrose as a cryoprotectant, then store the lyophilized powder at -20°C or 4°C with desiccant. Prior to use, reconstitute in the original volume of ultrapure water or appropriate buffer. To monitor antibody stability over time, periodically test aliquots on Western blots against a standardized positive control. Document storage conditions, aliquot dates, and freeze-thaw cycles for each batch to maintain quality control records. Using these approaches, antibodies typically retain activity for 3-5 years or longer.

How can protein complexes associated with At1g65740 be identified using antibody-based approaches?

Identifying protein complexes associated with At1g65740 can be achieved through several antibody-based approaches. Co-immunoprecipitation (Co-IP) is the primary method, where protein extracts are incubated with At1g65740 antibody to pull down the target protein along with its interacting partners . For transient interactions, implement crosslinking with formaldehyde (1%) or DSP (2 mM) prior to cell lysis. The immunoprecipitated complexes can be analyzed by mass spectrometry to identify components. For validation, perform reciprocal Co-IPs using antibodies against identified interactors. Another approach is proximity-dependent biotin identification (BioID), where a biotin ligase is fused to At1g65740, resulting in biotinylation of proximal proteins that can be purified using streptavidin and identified by mass spectrometry. Additionally, employ chromatin immunoprecipitation (ChIP) if At1g65740 is suspected to interact with DNA, to identify both protein-DNA and protein-protein interactions at chromatin. For visualizing protein complexes in situ, use proximity ligation assay (PLA), which produces fluorescent signals only when two proteins are in close proximity (<40 nm). These complementary methods provide a comprehensive view of the protein's interaction network in different cellular contexts.

How can At1g65740 antibody be used to study plant responses to biotic stressors?

At1g65740 antibody provides valuable tools for investigating plant responses to biotic stressors like pathogenic bacteria and fungi. First, use immunoblotting to track protein abundance changes during pathogen infection, comparing susceptible and resistant interactions. Plant immune responses involve dramatic transcriptional reprogramming , which likely affects protein expression patterns. Second, employ immunoprecipitation followed by mass spectrometry to identify stress-specific interaction partners that may function in immune signaling cascades. Plant immunity functions through complex receptor systems and signaling networks , where protein interactions are critical. Third, use immunolocalization to visualize protein translocation events during infection, as subcellular redistribution often occurs during immune responses. Fourth, combine antibody detection with genetic approaches using pathogen effector delivery systems to understand how specific virulence factors like HopAM1 might affect your protein of interest. Fifth, apply the antibody in chromatin immunoprecipitation studies if the protein has DNA-binding capabilities to identify stress-responsive genomic targets. Finally, use time-course experiments to map the temporal dynamics of protein abundance, modification, and localization during the progression of infection, providing insights into its functional role in plant immunity.

What are the key considerations for using At1g65740 antibody in ELISA-based quantification?

For reliable ELISA-based quantification using At1g65740 antibody, several critical factors must be addressed. First, optimize antibody concentration through checkerboard titration, testing primary antibody dilutions (1:500 to 1:10,000) against varying antigen concentrations to determine the optimal signal-to-noise ratio. Second, establish a standard curve using purified recombinant protein in the range of 0.1-100 ng/ml, fitting to a four-parameter logistic curve model for accurate quantification. Third, determine the assay's limit of detection (LOD) and limit of quantification (LOQ) by analyzing multiple blank samples and calculating mean + 3SD (LOD) and mean + 10SD (LOQ). Fourth, validate assay specificity using extracts from knockout plants and competition assays with purified antigen. Fifth, minimize matrix effects by preparing standards in the same buffer as samples or using sample dilution series to identify optimal working range. Sixth, address plate-to-plate variation by including calibration controls on each plate and normalizing results accordingly. Finally, implement quality control samples at low, medium, and high concentrations on each plate to monitor assay performance over time. Document these parameters thoroughly to ensure reproducibility across different experimental conditions and operators.

How can At1g65740 antibody be applied in the study of protein post-translational modifications?

At1g65740 antibody can be effectively employed to study post-translational modifications (PTMs) through multiple strategic approaches. First, use immunoprecipitation followed by mass spectrometry (IP-MS) to identify specific PTMs on the protein. Compare PTM profiles under different conditions (e.g., pathogen infection, abiotic stress) to identify regulatory modifications . Second, develop modification-specific antibodies that recognize At1g65740 only when modified in a particular way (e.g., phosphorylated, ubiquitinated). Third, perform two-dimensional Western blotting to separate protein isoforms based on charge (which is affected by many PTMs) before immunodetection. Fourth, use phosphatase or deubiquitinase treatments of immunoprecipitated protein to confirm the presence of phosphorylation or ubiquitination. Fifth, combine immunoprecipitation with specific PTM detection antibodies (anti-phospho, anti-ubiquitin) in sequential IPs or Western blots. Sixth, for temporal dynamics, implement pulse-chase experiments with immunoprecipitation at different time points to track modification turnover rates. Finally, verify the functional significance of identified PTMs by expressing modified versions of the protein (phosphomimetic or phospho-dead mutations) in knockout backgrounds and assessing phenotypic rescue. These approaches collectively provide comprehensive insights into how PTMs regulate protein function in various biological contexts.