At5g08505 Antibody

What is the At5g08505 protein and why is it studied?

At5g08505 is a defensin-like (DEFL) family protein encoded by the Arabidopsis thaliana genome. Defensin-like proteins are small cysteine-rich peptides that often play important roles in plant immune responses and development. These proteins are of interest to researchers studying plant defense mechanisms, stress responses, and cell signaling pathways. The At5g08505 protein has been identified and characterized through genomic and proteomic approaches, with its sequence cataloged in major databases with the UniProt accession number Q2V390 .

What are the key specifications of commercially available At5g08505 antibodies?

Commercial At5g08505 antibodies are typically polyclonal antibodies raised in rabbits against recombinant Arabidopsis thaliana At5g08505 protein. These antibodies are designed for research applications such as ELISA and Western blotting. The antibodies are typically supplied in liquid form in a storage buffer containing preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4). The antibodies are purified using antigen affinity methods to ensure specificity for the target protein .

How do I determine if the At5g08505 antibody will cross-react with orthologous proteins in other plant species?

When evaluating potential cross-reactivity, you should first perform sequence alignment analyses between At5g08505 and suspected orthologous proteins in your species of interest. Tools such as BLAST, Clustal Omega, or MUSCLE can help identify conserved epitope regions. Most commercial At5g08505 antibodies have been specifically tested for reactivity with Arabidopsis thaliana proteins, so cross-reactivity with other species must be empirically determined. Consider performing preliminary Western blot analyses with positive controls (Arabidopsis samples) alongside your species of interest. If cross-reactivity is suspected but weak, optimization of antibody concentration, incubation time, and detection methods may be necessary. Additionally, epitope mapping data, when available from the manufacturer, can help predict potential cross-reactivity based on sequence conservation .

How should I design experiments to validate At5g08505 antibody specificity in my system?

A comprehensive antibody validation strategy should include multiple approaches. Begin with Western blot analysis using wild-type Arabidopsis tissue extract alongside an At5g08505 knockout/knockdown line as a negative control. The antibody should detect a band of the expected molecular weight (~10 kDa for the mature protein) in wild-type samples but show reduced or absent signal in the knockout samples. For further validation, perform immunoprecipitation followed by mass spectrometry to confirm the identity of the captured protein. Consider using recombinant At5g08505 protein as a competitive inhibitor in your assay to demonstrate binding specificity. For immunolocalization studies, include appropriate blocking peptides and secondary antibody-only controls. Document all validation steps methodically, including antibody dilutions, incubation conditions, and detection methods used, as these parameters may need adjustment for different experimental contexts .

What are the optimal conditions for Western blot detection of At5g08505 protein?

For optimal Western blot detection of At5g08505 protein, begin with sample preparation by extracting proteins from Arabidopsis tissues using a buffer containing protease inhibitors to prevent degradation. Due to the small size of defensin-like proteins, use 15-20% SDS-PAGE gels for better resolution of low molecular weight proteins. After transfer to a PVDF or nitrocellulose membrane (PVDF is often preferred for small proteins), block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature. Incubate with the primary At5g08505 antibody at a 1:500 to 1:1000 dilution overnight at 4°C, followed by thorough washing with TBST. Apply an HRP-conjugated anti-rabbit secondary antibody at 1:5000 to 1:10000 dilution for 1 hour at room temperature. After washing, detect using an enhanced chemiluminescence system. For weak signals, consider signal amplification methods or extending the primary antibody incubation time. Optimization may be necessary based on protein expression levels and specific tissue types .

How can I effectively use the At5g08505 antibody in immunolocalization studies?

For effective immunolocalization of At5g08505 protein, tissue fixation and preparation are critical first steps. Fix Arabidopsis tissues in 4% paraformaldehyde for 2-4 hours, followed by dehydration and embedding in paraffin or resin. For whole-mount immunolocalization, fix tissues in 4% paraformaldehyde and permeabilize with a detergent such as Triton X-100. Block non-specific binding sites with 3-5% BSA or normal serum from the same species as the secondary antibody. Incubate with the At5g08505 primary antibody at a 1:100 to 1:200 dilution overnight at 4°C. After washing, apply fluorophore-conjugated secondary antibodies and counterstain with DAPI to visualize nuclei. Include controls such as samples incubated with pre-immune serum and secondary antibody-only treatments. For co-localization studies, ensure antibodies are raised in different species to avoid cross-reactivity. Confocal microscopy is recommended for optimal visualization, with special attention to defensin-like proteins' potential localization in cell walls, intercellular spaces, or secretory pathways .

What are the recommended storage conditions for maximizing At5g08505 antibody stability and shelf life?

To maximize stability and shelf life of At5g08505 antibodies, store them at -20°C or -80°C immediately upon receipt. The lower temperature (-80°C) is preferable for long-term storage beyond one year. Avoid repeated freeze-thaw cycles by aliquoting the antibody into smaller volumes (10-50 μl) based on typical usage amounts. Each aliquot should be in a sterile microcentrifuge tube with proper labeling including the antibody name, lot number, date of aliquoting, and concentration. For short-term storage (1-2 weeks), antibodies can be kept at 4°C. The antibody solution contains 50% glycerol and preservative (0.03% Proclin 300) to help maintain stability, but additional protectants like bovine serum albumin (BSA, 1-5 mg/ml) can be added if needed for diluted working solutions. Antibody performance should be periodically validated even under optimal storage conditions, particularly for critical experiments or after extended storage periods .

How should I prepare plant tissue samples to ensure optimal detection of At5g08505 protein?

Effective sample preparation is crucial for detecting defensin-like proteins such as At5g08505. Begin harvesting plant tissues at appropriate developmental stages and under relevant treatment conditions, as expression of defensin-like proteins often varies temporally and in response to stimuli. Flash-freeze harvested tissues immediately in liquid nitrogen and store at -80°C until processing. For protein extraction, grind tissues to a fine powder while maintaining frozen state, then extract using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and protease inhibitor cocktail. For secreted proteins, consider isolation of apoplastic fluid or culture media for cultured cells. Include reducing agents like DTT or β-mercaptoethanol in your sample buffer to properly denature the cysteine-rich defensin structure. Concentrate samples if necessary using protein precipitation methods (TCA/acetone) or centrifugal filters. Quantify protein concentration using Bradford or BCA assays and load equal amounts for comparative analyses. For recalcitrant tissues, optimization of extraction buffers may be necessary to overcome interference from secondary metabolites or cell wall components .

What quality control measures should I implement before using the At5g08505 antibody in critical experiments?

Implement a multi-step quality control protocol before using At5g08505 antibodies in critical experiments. First, verify physical appearance by inspecting for precipitates or contamination; any cloudiness or particles may indicate compromised quality. Perform a test Western blot using positive control samples (Arabidopsis thaliana tissue extract) and verify the appearance of a band at the expected molecular weight with minimal background. Assess batch-to-batch consistency by comparing current results with previous data if available. For quantitative applications, generate a standard curve using recombinant At5g08505 protein to confirm linear detection range. Cross-reactivity testing should be conducted by performing Western blots with extracts from knockout/knockdown lines or unrelated plant species. Document all quality control results in your laboratory records, including antibody dilution, incubation conditions, detection methods, and observed band patterns. For long-term studies, consider reserving a reference aliquot of validated antibody for future comparisons. If results are inconsistent, contact the manufacturer for technical support and possibly replacement .

How can I optimize antibody dilutions for different experimental applications?

Systematic titration is the key to optimizing At5g08505 antibody dilutions across different applications. For Western blotting, start with a dilution series (e.g., 1:250, 1:500, 1:1000, 1:2000) against a constant amount of positive control sample. Evaluate signal-to-noise ratio, band specificity, and background for each dilution. For ELISA, perform a checkerboard titration with both antigen and antibody dilution series to determine optimal concentrations yielding maximal signal difference between positive and negative samples while maintaining low background. For immunofluorescence or immunohistochemistry, begin with manufacturer-recommended dilutions (typically 1:100 to 1:200) and adjust based on signal intensity and background. Remember that optimal dilutions may vary between tissue types and preparation methods. Document all optimization results methodically, including incubation times, temperatures, and detection reagents used. When comparing results across experiments, maintain consistency in antibody lot numbers whenever possible. For applications requiring absolute quantification, include standard curves using purified recombinant At5g08505 protein to calibrate signals across different antibody dilutions .

What are the most common issues encountered with At5g08505 antibody experiments and how can they be resolved?

Several common issues may arise when working with At5g08505 antibodies. For weak or absent signals, consider: increasing antibody concentration, extending incubation time, using more sensitive detection systems, or optimizing protein extraction methods for defensin-like proteins. High background can be addressed by: increasing blocking agent concentration (5-10% BSA or milk), extending blocking time, adding 0.1-0.3% Tween-20 to wash buffers, or using more stringent washing procedures. Non-specific bands may appear due to: cross-reactivity with related proteins, sample degradation, or suboptimal SDS-PAGE conditions. To resolve, try increasing gel percentage (15-20%) for better resolution of small proteins, adding protease inhibitors during extraction, and performing appropriate negative controls. For inconsistent results between experiments, establish standardized protocols for sample preparation, use consistent antibody lots, and include positive controls in each experiment. If multiple detection methods produce conflicting results, consider protein confirmation by mass spectrometry. For defensin-like proteins specifically, optimizing reducing conditions in sample buffers may be necessary to properly denature their cysteine-rich structures for consistent epitope exposure .

How should I approach troubleshooting when my At5g08505 Western blot shows multiple unexpected bands?

When your Western blot shows multiple unexpected bands, follow a systematic troubleshooting approach. First, verify the predicted molecular weight of At5g08505 protein (~10 kDa for the mature protein, possibly larger if including signal peptide or post-translational modifications). Consider that defensin-like proteins may form dimers or multimers resistant to denaturation, resulting in higher molecular weight bands. Examine sample preparation: insufficient denaturation can be addressed by increasing SDS concentration, extending boiling time, or adding stronger reducing agents like DTT. For degradation products (bands smaller than expected), add fresh protease inhibitors during extraction and minimize sample processing time. To determine specificity, perform peptide competition assays using recombinant At5g08505 protein, which should reduce or eliminate specific bands. Compare patterns between different tissue types and growth conditions, as expression and modification may vary. For suspected cross-reactivity, test the antibody against At5g08505 knockout/knockdown samples. If high molecular weight bands persist, consider potential post-translational modifications such as glycosylation or ubiquitination, which can be verified with specific enzymatic treatments prior to Western blotting. Document all troubleshooting steps methodically for future reference .

How can I use the At5g08505 antibody to investigate protein-protein interactions in plant defense responses?

To investigate protein-protein interactions involving At5g08505, employ a multi-technique approach. Co-immunoprecipitation (Co-IP) serves as a foundational method: use the At5g08505 antibody to pull down the protein complex from plant extracts treated with appropriate crosslinking agents, followed by mass spectrometry to identify interacting partners. For validation of identified interactions, perform reciprocal Co-IPs using antibodies against the putative interacting proteins. Proximity ligation assays (PLA) can visualize interactions in situ by generating fluorescent signals when two proteins are within 40nm of each other. For monitoring dynamics of interactions during defense responses, extract proteins from plants at different time points after pathogen challenge or defense elicitor treatment. Bimolecular Fluorescence Complementation (BiFC) provides another validation approach by expressing At5g08505 and candidate interactors as fusion proteins with complementary fragments of fluorescent proteins. For defensin-like proteins specifically, consider their potential localization in extracellular spaces when designing extraction buffers and experimental conditions. Yeast two-hybrid screening can identify additional interactors, though results should be validated in planta due to potential differences in post-translational modifications between yeast and plant systems .

What approaches can I use to study post-translational modifications of the At5g08505 protein?

Studying post-translational modifications (PTMs) of At5g08505 requires targeted analytical approaches. Begin with immunoprecipitation using the At5g08505 antibody followed by mass spectrometry (MS) analysis, which can identify various PTMs including phosphorylation, glycosylation, and disulfide bond formation (particularly important for cysteine-rich defensins). For phosphorylation studies, use phospho-specific protein stains like Pro-Q Diamond or phospho-enrichment methods (TiO2 or IMAC) prior to MS analysis. Western blotting with modification-specific antibodies (anti-phospho, anti-ubiquitin, etc.) can complement MS findings. To identify functional impacts of PTMs, perform site-directed mutagenesis of modified residues followed by functional assays. For studying dynamic changes in PTMs during development or stress responses, conduct time-course experiments with appropriate treatments. Differential mobility on SDS-PAGE before and after treatment with specific enzymes (e.g., phosphatases, glycosidases) can provide initial evidence for modifications. For disulfide bonding patterns common in defensins, compare protein mobility under reducing versus non-reducing conditions. Consider advanced techniques like hydrogen-deuterium exchange mass spectrometry (HDX-MS) to assess how PTMs affect protein conformation and interaction surfaces .

How can I integrate At5g08505 antibody-based studies with transcriptomic and proteomic approaches?

Integrating At5g08505 antibody-based studies with -omics approaches creates a comprehensive understanding of this defensin-like protein's function. Start by correlating protein expression levels determined by Western blot quantification with mRNA expression data from RNA-seq or microarray studies across different tissues, developmental stages, or stress conditions. This correlation (or lack thereof) can reveal post-transcriptional regulation mechanisms. For spatial context, combine immunolocalization data with cell-type-specific transcriptomics to determine if At5g08505 protein localization matches gene expression patterns. Perform immunoprecipitation followed by mass spectrometry (IP-MS) to identify protein interactors, then cross-reference these with co-expression networks from transcriptomic data to identify functionally related genes. For regulatory studies, correlate At5g08505 protein levels with transcription factor binding data from ChIP-seq experiments. In stress response studies, use the antibody to track protein abundance changes and compare with temporal transcriptome data to establish response kinetics. Utilize targeted proteomics (selected reaction monitoring) with isotopically labeled peptide standards for absolute quantification of At5g08505 protein, enabling precise stoichiometric comparisons with interacting proteins. For functional genomics approaches, compare antibody-detected protein levels in wild-type plants versus mutant lines to connect genotype with protein phenotype .

What statistical approaches are most appropriate for quantifying At5g08505 protein levels across different experimental conditions?

For rigorous quantification of At5g08505 protein levels, employ appropriate statistical methods based on your experimental design. For Western blot densitometry, normalize band intensities to loading controls (such as actin or GAPDH) before statistical analysis. When comparing multiple treatment groups, use ANOVA followed by post-hoc tests (Tukey's HSD or Dunnett's test when comparing to a control group) rather than multiple t-tests to control for family-wise error rate. For non-normally distributed data, apply non-parametric alternatives such as Kruskal-Wallis followed by Dunn's test. Perform power analysis before experiments to determine appropriate sample sizes (typically n≥3 biological replicates, each with 2-3 technical replicates). For time-course experiments, consider repeated measures ANOVA or mixed-effects models to account for within-subject correlations. When analyzing ELISA data, use log-transformation if necessary to achieve normality and homoscedasticity. Report effect sizes (Cohen's d or fold change) alongside p-values to indicate biological significance. For complex experimental designs with multiple factors (e.g., genotype, treatment, time), use factorial ANOVA or general linear models to assess main effects and interactions. Provide clear visualization of data using box plots or dot plots showing individual data points rather than bar graphs with error bars alone .

How do I interpret conflicting results between antibody-based detection and transcriptomic data for At5g08505?

Discrepancies between protein detection and transcriptomic data for At5g08505 may reveal important biological insights rather than experimental errors. First, verify technical aspects: confirm antibody specificity using appropriate controls and check RNA quality and primer specificity for transcriptomic data. Consider temporal dynamics—protein levels often lag behind transcript changes, so time-course sampling may resolve apparent conflicts. Post-transcriptional regulation mechanisms should be investigated, including miRNA-mediated degradation, RNA binding proteins affecting stability, or translational control. Assess protein stability factors such as ubiquitination or other degradation signals that might lead to rapid protein turnover despite high transcript levels. For defensin-like proteins specifically, examine possible protein secretion that could result in low intracellular protein levels despite high transcript expression. Differential extraction efficiency could also explain discrepancies if the protein is tightly bound to cell walls or membranes. Employ polysome profiling to determine if transcripts are actively translated. Integrate multiple detection methods; for example, supplement Western blotting with immunofluorescence to assess protein localization. Remember that transcript-protein correlation varies widely among genes, with regulatory and signaling proteins often showing poorer correlation than structural or metabolic proteins .

How can I determine whether observed changes in At5g08505 protein levels are physiologically significant?

Determining physiological significance of At5g08505 protein level changes requires multiple lines of evidence beyond statistical significance. Begin by establishing dose-response relationships between protein levels and observed phenotypes using genetic approaches such as overexpression and knockdown/knockout lines with varying expression levels. Compare observed changes against natural variation in wild-type plants across different tissues, developmental stages, and environmental conditions to determine if experimental changes exceed normal fluctuation ranges. For defensin-like proteins, which often function in stress responses, challenge plants with relevant stressors and assess correlation between protein levels and stress tolerance phenotypes. Conduct target validation experiments using recombinant At5g08505 protein in appropriate bioassays (e.g., antimicrobial activity for defensins) at concentrations matching those observed in planta. Integrate protein-level changes with metabolomic data to identify downstream effects on relevant metabolic pathways. For potentially pleiotropic effects, use tissue-specific or inducible expression systems to manipulate At5g08505 levels in specific contexts. Time-course studies can reveal whether protein level changes precede or follow physiological responses, helping establish causality versus correlation. Consider evolutionary conservation by examining orthologous proteins in related species and their functional significance. Document all phenotypic changes comprehensively, including subtle effects on growth, development, and stress responses that might be overlooked in focused studies .

Research Applications Table