At5g64970 Antibody

What is the At5g64970 Antibody and what protein does it target?

The At5g64970 Antibody (Product Code: CSB-PA172142XA01DOA) is a primary antibody specifically designed to detect and bind to the protein encoded by the At5g64970 gene in Arabidopsis thaliana . This gene encodes a protein with UniProt accession number Q9LV81, which is involved in specific cellular processes in this model plant organism . The antibody serves as a critical tool for researchers investigating protein expression, localization, and function related to this specific gene product. Unlike general-purpose antibodies, the At5g64970 Antibody offers high specificity for its target protein, making it valuable for precise detection in complex plant tissue samples and cellular extracts.

What are the technical specifications of the At5g64970 Antibody?

The At5g64970 Antibody is available in two size options: 2ml and 0.1ml quantities . The antibody has been validated specifically for Arabidopsis thaliana applications, with confirmed reactivity against the Q9LV81 protein . This primary antibody is designed for various research applications including Western blotting, immunohistochemistry, ELISA, and possibly immunofluorescence, depending on the specific validation tests performed by the manufacturer. When selecting this antibody for research purposes, it's important to consider the concentration, formulation buffer, and storage conditions to ensure optimal antibody performance and longevity. Most primary antibodies of this type require storage at -20°C and have specific dilution recommendations for different experimental applications.

What information is available about the At5g64970 gene and its protein product?

The At5g64970 gene in Arabidopsis thaliana encodes a protein with UniProt accession number Q9LV81 . This protein plays specific roles in plant cellular processes, though detailed functional characterization may still be emerging in current research. Understanding the protein's molecular weight, isoelectric point, known domains, post-translational modifications, and cellular localization is crucial for designing appropriate experimental conditions when using this antibody. Researchers should consult current literature and protein databases for the most up-to-date functional annotations of the At5g64970 gene product to properly interpret antibody-based experimental results and place them in the appropriate biological context.

How should I optimize Western blot protocols when using At5g64970 Antibody?

When optimizing Western blot protocols with At5g64970 Antibody, begin with protein extraction using a plant-specific buffer containing protease inhibitors to prevent degradation of the target protein. For Arabidopsis samples, a standard extraction ratio is 100mg tissue per 300μl buffer. Determine the appropriate protein amount (typically 20-40μg per lane) through preliminary experiments. The optimal antibody dilution generally ranges from 1:500 to 1:2000, but specific titration is recommended. Include positive controls (known At5g64970-expressing samples) and negative controls (knockout lines if available) to validate specificity. Blocking solutions based on 5% non-fat milk or BSA in TBST are typically effective, with overnight primary antibody incubation at 4°C. If background is excessive, increase washing steps (5x 10 minutes with TBST) and optimize secondary antibody dilution (typically 1:5000 to 1:10000). For enhanced sensitivity with low abundance proteins, consider using chemiluminescent detection systems with extended exposure times.

Which sample preparation methods yield the best results for At5g64970 detection in Arabidopsis tissues?

The most effective sample preparation for At5g64970 detection begins with proper tissue collection and preservation. Harvest Arabidopsis tissues at appropriate developmental stages, flash-freeze in liquid nitrogen, and store at -80°C until processing. For protein extraction, use a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and a complete protease inhibitor cocktail. Mechanical disruption methods such as bead-beating or mortar-and-pestle grinding under liquid nitrogen yield superior protein recovery compared to chemical lysis alone. For subcellular fractionation studies, differential centrifugation protocols should be optimized for the predicted cellular location of the At5g64970 protein product. When working with specific plant tissues (roots, leaves, flowers), tissue-specific extraction modifications may be necessary to address varying levels of interfering compounds like polyphenols or polysaccharides. For challenging tissues, additional purification steps such as acetone precipitation or TCA/acetone extraction may improve detection sensitivity by removing interfering compounds.

What are the recommended procedures for immunohistochemistry using At5g64970 Antibody?

For immunohistochemistry with At5g64970 Antibody, first fix Arabidopsis tissues in 4% paraformaldehyde for 12-24 hours, followed by paraffin embedding and sectioning at 5-8μm thickness. Deparaffinize sections completely and perform antigen retrieval using citrate buffer (pH 6.0) at 95°C for 20-30 minutes to expose epitopes that may have been masked during fixation. Block endogenous peroxidase activity with 3% hydrogen peroxide and prevent non-specific binding with 5% normal serum from the species in which the secondary antibody was raised. Apply At5g64970 Antibody at a 1:50 to 1:200 dilution (to be optimized) and incubate overnight at 4°C in a humidified chamber. For detection, use a compatible secondary antibody system, either fluorescent or enzyme-based (such as HRP), depending on the desired visualization method. Include controls for autofluorescence, which is common in plant tissues, and use appropriate counterstains to provide tissue context. Specific plant tissue clearing techniques may be necessary for whole-mount immunohistochemistry applications to improve antibody penetration and reduce background fluorescence from chlorophyll and other plant pigments.

How can I validate the specificity of At5g64970 Antibody in my experimental system?

Validating At5g64970 Antibody specificity requires multiple complementary approaches. First, perform Western blot analysis comparing wild-type Arabidopsis with at5g64970 knockout or knockdown lines, expecting signal reduction or absence in the mutant. Preabsorption tests, where the antibody is pre-incubated with purified target protein before application to samples, should abolish specific signals if the antibody is truly specific. Mass spectrometry analysis of immunoprecipitated material can confirm the identity of the detected protein. For genetic validation, complement mutant lines with the At5g64970 gene and confirm restored antibody signal. Cross-reactivity testing with closely related proteins, particularly in heterologous expression systems, can establish binding specificity. When possible, employ orthogonal detection methods such as RNA expression analysis (qPCR or RNA-seq) to correlate protein detection with transcript levels. Document all validation steps meticulously, as antibody performance can vary between experimental conditions and even between antibody lots from the same manufacturer.

What approaches should I use for troubleshooting weak or absent signals when using At5g64970 Antibody?

When encountering weak or absent signals with At5g64970 Antibody, implement a systematic troubleshooting strategy. First, confirm protein extraction efficiency using total protein stains like Ponceau S on membranes. Validate sample integrity by detecting abundant housekeeping proteins with established antibodies. For Western blot applications, increase protein loading (50-100μg), reduce antibody dilution (1:250-1:500), extend primary antibody incubation (overnight at 4°C or 48 hours), or switch to more sensitive detection systems (enhanced chemiluminescence or fluorescent secondary antibodies). Consider alternative antigen retrieval methods for immunohistochemistry, such as enzymatic digestion with proteinase K or trypsin. The At5g64970 protein may be expressed at low levels or in specific developmental stages or conditions; consult expression databases to determine optimal sampling conditions. If the protein undergoes rapid turnover, treat samples with proteasome inhibitors (MG132) prior to extraction. For membrane-associated proteins, optimize detergent types and concentrations (CHAPS, digitonin, or DDM as alternatives to Triton X-100). Test different antibody clones if available, as epitope accessibility may vary between antibodies targeting different regions of the same protein.

How can At5g64970 Antibody be utilized in co-immunoprecipitation studies to identify protein interaction partners?

For co-immunoprecipitation (co-IP) studies with At5g64970 Antibody, begin with optimized extraction conditions that preserve protein-protein interactions. Use mild lysis buffers containing 0.5-1% NP-40 or Digitonin rather than stronger detergents like SDS or deoxycholate. Pre-clear lysates with Protein A/G beads to reduce non-specific binding. Covalently cross-link the At5g64970 Antibody to Protein A/G beads using dimethyl pimelimidate (DMP) to prevent antibody co-elution and contamination of mass spectrometry samples. Include appropriate controls: IgG isotype control for non-specific binding, input samples for abundance normalization, and when possible, samples from at5g64970 mutant plants as negative controls. For plant tissues with abundant polyphenols or polysaccharides, include PVPP or specific additives in extraction buffers to reduce interference. Consider complementary approaches such as proximity-labeling methods (BioID or APEX) or yeast two-hybrid screens to validate interaction partners. For transient interactions, perform crosslinking with formaldehyde or DSP prior to cell lysis. When analyzing co-IP results, prioritize proteins enriched relative to both input and control IP samples, and validate key interactions through reciprocal co-IPs with antibodies against the putative interacting partners.

How should I design experiments to study protein expression changes across different plant developmental stages?

Designing experiments to study At5g64970 protein expression across developmental stages requires careful planning. Establish a comprehensive sampling timeline covering key developmental transitions in Arabidopsis (seedling, vegetative growth, flowering, senescence). Maintain uniform growing conditions (light, temperature, humidity) to minimize environmental variables. For each developmental stage, collect multiple biological replicates (minimum n=3) and process tissues consistently for protein extraction. Normalize protein loading using total protein measurement methods (Bradford assay) rather than housekeeping proteins, which may vary across developmental stages. Include well-characterized developmental markers as controls to validate stage-specific sampling. For quantitative analysis, consider using fluorescent secondary antibodies and digital imaging systems rather than chemiluminescence, as they provide better linearity for quantification. Validate protein expression patterns through complementary approaches such as promoter-reporter fusions or transcript analysis. Statistical analysis should account for biological variability using appropriate tests (ANOVA with post-hoc comparisons) to identify significant changes across developmental stages.

What controls are essential when performing subcellular localization studies using At5g64970 Antibody?

For subcellular localization studies with At5g64970 Antibody, multiple controls are essential for reliable interpretation. Include markers for relevant subcellular compartments (nucleus, chloroplast, mitochondria, ER, Golgi, vacuole, plasma membrane) to establish spatial references. Perform co-localization with fluorescently tagged organelle markers when using immunofluorescence techniques. Include appropriate negative controls: primary antibody omission, isotype control antibodies, and when available, tissues from at5g64970 knockout plants. For validation, compare antibody-based localization with fluorescently tagged At5g64970 protein expressed under native promoter control in transgenic plants. Be aware of potential artifacts from overexpression systems. When performing subcellular fractionation followed by Western blotting, include established marker proteins for each fraction to confirm separation quality. Assess potential cross-reactivity with similar proteins in the same subcellular compartment through immunoprecipitation followed by mass spectrometry. Document imaging parameters comprehensively (exposure times, gain settings), especially when performing quantitative comparisons between conditions, and use consistent parameters across experimental groups.

How can I effectively use At5g64970 Antibody in stress response studies in Arabidopsis?

For stress response studies utilizing At5g64970 Antibody, implement a systematic experimental design. First, establish appropriate stress conditions (drought, salt, heat, cold, pathogen) with graduated intensity levels and time points based on literature and preliminary experiments. Include proper controls for each stress condition: mock treatments, recovery periods, and positive controls using genes with well-characterized stress responses. Standardize tissue collection protocols (time of day, plant age, tissue type) to minimize variability. For short-term stress responses, consider kinetic sampling (15min, 30min, 1h, 3h, 6h, 24h) to capture transient changes in protein levels. When comparing protein levels across stress conditions, use consistent protein extraction methods and loading controls. Consider post-translational modifications that may occur during stress responses by analyzing migration patterns carefully and potentially using phospho-specific antibodies if relevant. Correlate protein expression data with physiological measurements of stress (relative water content, electrolyte leakage, photosynthetic efficiency) to establish functional relevance. Validate key findings in multiple Arabidopsis ecotypes to ensure the response is not ecotype-specific. Complement antibody-based analysis with transcript level measurements to distinguish between transcriptional and post-transcriptional regulation mechanisms.

What statistical approaches are recommended for quantifying Western blot data with At5g64970 Antibody?

For quantitative analysis of Western blot data using At5g64970 Antibody, employ rigorous statistical approaches beginning with proper experimental design. Perform a minimum of three independent biological replicates, each with technical duplicates when possible. For densitometry analysis, use dedicated software (ImageJ, Image Lab, etc.) with consistent quantification parameters. Normalize target protein signal to an appropriate loading control or total protein stain (Ponceau S, SYPRO Ruby) rather than single housekeeping proteins, which may vary under experimental conditions. Test data for normality using Shapiro-Wilk or Kolmogorov-Smirnov tests before selecting parametric (t-test, ANOVA) or non-parametric (Mann-Whitney, Kruskal-Wallis) statistical tests. For multiple comparisons, apply appropriate correction methods (Bonferroni, Tukey, or false discovery rate). Report not only p-values but also effect sizes and confidence intervals to indicate biological significance beyond statistical significance. For complex experimental designs (multiple treatments, time points, or genotypes), consider two-way or three-way ANOVA followed by appropriate post-hoc tests. Present quantitative Western blot data with both representative images and quantification graphs showing individual data points, means, and error bars representing standard deviation or standard error as appropriate.

How should I address potential cross-reactivity issues when interpreting results?

Addressing cross-reactivity concerns with At5g64970 Antibody requires systematic analytical approaches. First, examine Western blot results for unexpected bands that may indicate binding to proteins other than the intended target. Consult protein databases to identify Arabidopsis proteins with similar molecular weights and/or sequence homology to the At5g64970 protein. Verify specificity using genetic approaches by comparing wild-type plants with at5g64970 knockout or knockdown lines; specific bands should be reduced or absent in mutant samples. For ambiguous results, perform immunoprecipitation followed by mass spectrometry to identify all proteins recognized by the antibody. If cross-reactivity is detected, implement additional validation steps such as using a second antibody targeting a different epitope of the same protein or complementary techniques like RNA expression analysis. When reporting results, clearly document all bands observed, not just those at the expected molecular weight, and discuss potential cross-reactivity limitations. If significant cross-reactivity is unavoidable, consider developing alternative approaches such as epitope-tagged transgenic lines or generating more specific antibodies against unique regions of the At5g64970 protein.

How can I integrate antibody-based data with transcriptomic and proteomic datasets?

Integrating At5g64970 Antibody-derived data with transcriptomic and proteomic datasets requires methodical multi-omics approaches. Begin by ensuring experimental conditions are comparable across different data types, ideally using samples from the same biological material. For correlation analysis between protein levels (antibody detection) and transcript levels (RNA-seq or microarray), normalize each dataset appropriately before calculating Pearson or Spearman correlation coefficients. Be aware that correlation may be imperfect due to post-transcriptional regulation, protein stability differences, or temporal delays between transcription and translation. When comparing antibody-based detection with mass spectrometry-based proteomics, consider differences in detection sensitivity and dynamic range between methods. Use pathway analysis tools (GO enrichment, KEGG pathway mapping) to place At5g64970 in its biological context based on co-expressed genes or co-regulated proteins. Develop network models incorporating protein-protein interaction data from co-IP experiments with transcriptional regulatory information. For visualization, use integrated multi-omics visualization tools (Cytoscape, MapMan) to create comprehensive models showing relationships between transcript levels, protein abundance, and functional networks. When discrepancies arise between data types, investigate potential biological explanations such as post-translational modifications, protein localization changes, or context-dependent regulation rather than simply attributing differences to technical variations.

What novel applications of At5g64970 Antibody are emerging in plant research?

Emerging applications of At5g64970 Antibody include advanced spatial biology techniques that provide unprecedented insights into protein localization and dynamics. Super-resolution microscopy methods (STORM, PALM, SIM) are increasingly being applied with plant antibodies to visualize protein distribution at nanometer resolution, overcoming the diffraction limit of conventional microscopy. Combining At5g64970 Antibody with expansion microscopy, where tissues are physically expanded while maintaining relative protein positions, allows visualization of protein complexes previously unresolvable in dense plant tissues. In situ proximity ligation assays (PLA) using the At5g64970 Antibody paired with antibodies against putative interacting partners can visualize protein-protein interactions directly within cellular contexts. Single-cell proteomics approaches are beginning to integrate antibody-based detection methods with microfluidic systems to analyze protein expression heterogeneity across different cell types within plant tissues. Additionally, researchers are developing antibody-based biosensors by conjugating At5g64970 Antibody with environmentally-sensitive fluorophores that change properties upon binding, allowing real-time monitoring of protein dynamics in living plant systems.

How can I adapt chromatin immunoprecipitation (ChIP) protocols for use with At5g64970 Antibody?

Adapting chromatin immunoprecipitation (ChIP) protocols for At5g64970 Antibody requires several plant-specific modifications. Begin with proper crosslinking using 1-3% formaldehyde for 10-15 minutes under vacuum infiltration to ensure penetration through plant cell walls. Include a glycine quenching step (125mM final concentration) to stop crosslinking. For chromatin extraction, use a plant-specific nuclear isolation buffer containing β-mercaptoethanol, protease inhibitors, and plant nuclear isolation aids such as Triton X-100. Sonication parameters require careful optimization for plant tissues; typically, 10-15 cycles of 30 seconds on/30 seconds off at medium power achieves chromatin fragments of 200-500bp. Pre-clear chromatin with protein A/G beads and non-specific IgG to reduce background. For immunoprecipitation, use 3-5μg of At5g64970 Antibody per ChIP reaction and incubate overnight at 4°C with rotation. Include appropriate controls: input chromatin (pre-IP sample), IgG control IP, and positive control IP using antibodies against well-characterized chromatin-associated proteins like histones. After reverse crosslinking and DNA purification, verify enrichment using qPCR for predicted binding regions before proceeding to genome-wide analyses like ChIP-seq. For plant ChIP-seq, typically 10-20 million sequencing reads are recommended for adequate coverage.

What considerations are important when using At5g64970 Antibody in high-throughput screening approaches?

When implementing At5g64970 Antibody in high-throughput screening, several critical considerations ensure reliable results. First, perform rigorous antibody validation in the specific assay format being used for screening, as antibody performance can differ between applications. Develop robust positive and negative controls that can be included on every assay plate to monitor assay performance and facilitate normalization. For automated immunoassays, optimize antibody concentration through careful titration experiments to determine the minimum effective concentration that provides adequate signal-to-noise ratio, conserving valuable antibody resources. Implement quality control metrics such as Z'-factor calculations to assess assay robustness, aiming for Z' values >0.5. Minimize edge effects on multi-well plates by using buffer-filled perimeter wells or humidified incubation chambers. For fluorescence-based detection, address plant autofluorescence through appropriate filter sets and background subtraction algorithms. When screening plant compound libraries, include controls for potential interference from pigments, polyphenols, or other phytochemicals that might affect antibody binding or detection systems. Develop standardized data analysis pipelines that include normalization steps, outlier detection methods, and hit selection criteria appropriate for the biological question being addressed. For validation of screening hits, include orthogonal secondary assays that use different detection principles to confirm the biological relevance of identified candidates.

How does At5g64970 Antibody compare to other antibodies targeting related Arabidopsis proteins?

The At5g64970 Antibody exhibits distinct characteristics when compared to antibodies targeting related Arabidopsis proteins. Based on available data, this antibody demonstrates high specificity for its target protein (UniProt: Q9LV81) , with minimal cross-reactivity to other plant proteins sharing similar domains. Unlike antibodies targeting highly conserved proteins such as CAM (calmodulin) family members, which often show cross-reactivity across multiple isoforms, the At5g64970 Antibody offers more selective detection capabilities. When compared to antibodies against other Arabidopsis proteins listed in the catalog, such as BRN1 (Q8LFS6) or BRK1 (Q94JY4), the At5g64970 Antibody likely has distinct epitope recognition properties based on the unique sequence regions of its target protein . Researchers should consider these specificity differences when designing multiplexed experiments involving detection of multiple related proteins. Performance comparisons across different applications (Western blot vs. immunohistochemistry) may vary between antibodies, necessitating application-specific validation for each antibody rather than assuming uniform performance across all experimental platforms.

How is At5g64970 Antibody being used in current plant biology research?

At5g64970 Antibody is currently being utilized in various cutting-edge research areas in plant biology. Researchers are employing this antibody in studies investigating protein-protein interaction networks in Arabidopsis, particularly in contexts where the At5g64970 protein product may participate in signaling pathways or molecular complexes. The antibody has applications in developmental biology research, where tracking protein expression across different tissues and growth stages provides insights into the temporal and spatial regulation of At5g64970. In stress biology research, this antibody helps investigate how environmental challenges alter expression or post-translational modifications of the target protein. When combined with CRISPR/Cas9 gene editing approaches, the antibody serves as a validation tool to confirm successful protein knockdown or modification in mutant lines. Plant synthetic biology projects may utilize this antibody to verify successful heterologous expression or protein engineering involving the At5g64970 gene product. As multi-omics approaches become increasingly common, the antibody provides protein-level validation for transcriptomic and metabolomic findings, creating integrated models of plant cellular processes involving the At5g64970 protein and its interacting partners.

What emerging technologies might enhance the utility of At5g64970 Antibody in future research?

Emerging technologies are poised to significantly expand the research applications of At5g64970 Antibody. Microfluidic antibody arrays will enable simultaneous detection of At5g64970 alongside dozens of other proteins in minimal sample volumes, allowing comprehensive pathway analysis from limited plant material. Advances in single-cell proteomics will extend antibody-based detection to the single-cell level, revealing cell-type-specific expression patterns previously masked in whole-tissue analyses. Mass cytometry (CyTOF) using metal-conjugated antibodies will allow multiplexed protein detection without fluorescence spectrum limitations, enabling comprehensive protein network analysis in plant systems. The integration of CRISPR screening technologies with antibody-based detection methods will facilitate high-throughput functional genomics studies correlating genetic perturbations with At5g64970 protein expression changes. Computational advances in structural biology may improve epitope prediction, enabling development of next-generation antibodies with enhanced specificity and affinity. Nanotechnology-based approaches, such as quantum dot-conjugated antibodies, will provide enhanced sensitivity and photostability for long-term imaging experiments. Additionally, developments in tissue clearing and 3D imaging technologies will allow whole-organ imaging with cellular resolution, mapping At5g64970 distribution throughout intact plant structures rather than in thin sections.