At1g54445 Antibody

What is At1g54445 antibody and what is its specificity profile?

At1g54445 antibody (CSB-PA647281XA01DOA) is a custom antibody designed to detect the protein product of the At1g54445 gene in Arabidopsis thaliana (Mouse-ear cress). This antibody specifically targets the Q2V4G9 protein, enabling researchers to investigate its expression and function in plant tissues . The antibody's specificity is established through rigorous validation procedures including western blotting, immunoprecipitation, and immunohistochemistry to ensure minimal cross-reactivity with other proteins within the experimental system. The antibody recognizes specific epitopes (antigenic determinants) on the target protein, allowing for precise identification and quantification in experimental contexts. Compared to other antibodies targeting Arabidopsis proteins, such as SCRL and LCR antibodies listed in the catalog, At1g54445 antibody maintains its own distinct specificity profile optimized for its target protein .

How should I select appropriate controls when using At1g54445 antibody in my experiments?

Selecting appropriate controls is critical for experimental validity when working with At1g54445 antibody. For positive controls, consider using Arabidopsis thaliana tissue samples known to express the target protein, ideally verified through independent methods such as RT-PCR or RNA-seq data. Negative controls should include tissues where the target protein is absent or tissues from knockout/knockdown lines of the At1g54445 gene. Additionally, implement technical negative controls by omitting the primary antibody in your protocol while maintaining all other reagents and procedures .

When designing experiments, include isotype controls matching the At1g54445 antibody's host species and immunoglobulin class but lacking specific target recognition. This controls for potential non-specific binding. For more sophisticated experiments, consider using secondary antibody-only controls and pre-absorption controls where the antibody is pre-incubated with purified target antigen before application to the sample . These comprehensive controls help distinguish between specific signal and background noise, increasing confidence in your results and addressing potential experimental biases.

What are the recommended storage and handling protocols for At1g54445 antibody?

To maintain optimal activity of At1g54445 antibody, follow these evidence-based handling protocols: Store the antibody at -20°C for long-term storage and at 4°C for short-term use (up to one month). The antibody is typically available in two sizes: 2ml or 0.1ml, with appropriate storage volumes proportionate to concentration . Avoid repeated freeze-thaw cycles by aliquoting the antibody into single-use volumes upon receipt—typically 10-50μl depending on your experimental needs—using sterile, low-protein binding microcentrifuge tubes.

During handling, maintain aseptic conditions and use sterile pipette tips. When diluting the antibody for experimental use, use high-quality buffers free of microbial contamination. Typical working dilutions range from 1:500 to 1:2000 depending on the application (e.g., Western blotting vs. immunohistochemistry), but optimal dilution should be determined empirically for each experimental system. Document lot numbers, receipt dates, and remaining volumes to ensure experimental reproducibility and proper inventory management. Following these methodological practices ensures maximum antibody performance and experimental reliability across multiple research projects.

How should I design experiments to validate At1g54445 antibody specificity in my particular research model?

To validate At1g54445 antibody specificity in your research model, implement a systematic experimental design approach. Begin with western blot analysis using whole-cell lysates from Arabidopsis thaliana tissue expressing the target protein, looking for a single band at the expected molecular weight of the Q2V4G9 protein. Compare wild-type samples with knockout/knockdown lines of At1g54445 gene to confirm signal disappearance or reduction .

Next, employ immunoprecipitation followed by mass spectrometry to independently confirm that the antibody pulls down the intended target. For spatial validation, conduct immunohistochemistry or immunofluorescence experiments comparing the staining pattern with known expression data from transcriptomic studies. To control for potential cross-reactivity, perform heterologous expression experiments by expressing the At1g54445 protein in a non-plant system and confirming antibody recognition .

Design your validation experiments with appropriate statistical power by including sufficient biological and technical replicates (minimum n=3 for each). Document all experimental parameters meticulously, including antibody dilution, incubation times and temperatures, buffer compositions, and detection methods. This comprehensive validation approach ensures that your subsequent experimental findings using this antibody will be both reliable and reproducible across different experimental conditions.

What experimental design considerations are important when using At1g54445 antibody in quantitative analyses?

When designing quantitative experiments with At1g54445 antibody, several critical factors must be addressed. First, establish a standard curve using purified recombinant Q2V4G9 protein to determine the linear dynamic range of detection. This allows accurate quantification without signal saturation or falling below detection limits . Implement a randomized block design to control for potential batch effects, particularly when processing multiple samples across different experimental days.

For quantitative western blotting, include an invariant loading control protein and use normalization to account for lane-to-lane variations. When conducting ELISA or similar immunoassays, run all standards and samples in triplicate to enable statistical assessment of measurement precision. The experimental design should include both technical replicates (repeated measurements of the same sample) and biological replicates (independent biological samples) to distinguish between measurement variability and true biological variation .

How can I design experiments to investigate protein-protein interactions involving the At1g54445 protein?

To investigate protein-protein interactions involving At1g54445 protein, design a multi-method experimental approach that provides complementary lines of evidence. Begin with co-immunoprecipitation experiments using At1g54445 antibody to pull down the target protein along with its interaction partners from Arabidopsis thaliana lysates. Follow this with reverse co-immunoprecipitation using antibodies against suspected interaction partners . Analyze the precipitated complexes using mass spectrometry to identify novel interacting proteins.

For in vivo validation, design proximity ligation assays (PLA) or fluorescence resonance energy transfer (FRET) experiments to confirm interactions in their native cellular context. These techniques require careful control experiments including tests for non-specific antibody binding and autofluorescence measurements. When designing yeast two-hybrid or split-complementation assays as orthogonal validation methods, construct appropriate fusion proteins that preserve functional domains and protein folding .

Experimental controls should include testing interactions with known non-interacting proteins as negative controls and, where available, previously confirmed interaction partners as positive controls. For quantitative assessment of interaction strength, design surface plasmon resonance or isothermal titration calorimetry experiments using purified proteins. Throughout all interaction studies, maintain consistent experimental conditions (buffer composition, salt concentration, pH, temperature) that reflect the physiological environment in which these interactions naturally occur. This comprehensive experimental design strategy provides robust evidence for genuine protein-protein interactions involving the At1g54445 protein.

How should I analyze and interpret contradictory results when using At1g54445 antibody across different experimental platforms?

When facing contradictory results with At1g54445 antibody across different experimental platforms, implement a systematic analytical approach. First, evaluate the specificity of the antibody in each platform separately by reviewing your validation data, particularly comparing the detection pattern between wild-type and knockout samples. Contradictions often arise from platform-specific factors rather than the antibody itself .

Create a detailed comparison table documenting all experimental parameters across platforms:

Analyze each contradiction systematically, considering that different experimental conditions may expose different epitopes or affect antibody-antigen interactions. For instance, the At1g54445 protein may undergo post-translational modifications or conformational changes that affect antibody recognition in specific contexts .

Instead of dismissing contradictory results, design targeted experiments to resolve discrepancies, such as epitope mapping or testing whether the protein undergoes context-dependent modifications. Present both confirmatory and contradictory data in your research with appropriate statistical analyses, as this transparency enhances scientific rigor. Remember that contradictions often lead to new discoveries about protein behavior across different cellular contexts.

What statistical approaches are most appropriate for analyzing data generated using At1g54445 antibody?

When analyzing data generated using At1g54445 antibody, select statistical approaches that align with your experimental design and data characteristics. For quantitative western blot or ELISA data, first test for normality using Shapiro-Wilk or Kolmogorov-Smirnov tests before proceeding with parametric or non-parametric analyses . For comparisons between two experimental groups, apply Student's t-test for normally distributed data or Mann-Whitney U test for non-normally distributed data.

For experiments with multiple groups or conditions, implement one-way or two-way ANOVA with appropriate post-hoc tests (Tukey's HSD for all pairwise comparisons or Dunnett's test when comparing multiple groups to a control). Include power analysis calculations to ensure your sample size provides adequate statistical power (typically aiming for 0.8 or higher) .

For immunohistochemistry or immunofluorescence data, consider both intensity measurements and spatial distribution patterns. Employ image analysis software to quantify signal intensity in defined cellular compartments, followed by appropriate statistical tests. For colocalization studies, use Pearson's or Mander's correlation coefficients rather than visual assessment alone .

How can I properly interpret At1g54445 antibody data in relation to gene expression studies?

Integrating At1g54445 antibody data with gene expression studies requires careful methodological considerations to account for the different regulatory levels being measured. When comparing protein levels detected by At1g54445 antibody with mRNA expression data, remember that these represent distinct biological processes—transcription versus the combined effects of translation, post-translational modifications, and protein degradation .

First, establish appropriate time-course experiments that account for the temporal delay between transcription and observable protein expression. In Arabidopsis, this delay typically ranges from hours to days depending on the protein. When discrepancies arise between mRNA and protein levels, systematically investigate potential regulatory mechanisms such as microRNA regulation, differential translation efficiency, or protein stability differences .

Create correlation analyses between mRNA and protein data across multiple experimental conditions, calculating Spearman's rank correlation coefficients which can capture non-linear relationships. Present this data in scatter plots with regression lines to visualize the relationship strength. For more sophisticated analysis, implement mathematical models that incorporate parameters for translation rates and protein degradation, which can explain apparent discrepancies .

How can At1g54445 antibody be utilized in advanced plant stress response studies?

At1g54445 antibody offers sophisticated applications in plant stress response studies through multi-dimensional experimental approaches. Design time-course experiments exposing Arabidopsis thaliana to various abiotic stressors (drought, salt, temperature extremes) and biotic challenges (pathogen infection), followed by quantitative immunoblotting to track changes in At1g54445 protein levels, post-translational modifications, and subcellular localization .

Implement co-immunoprecipitation coupled with mass spectrometry to identify stress-induced changes in the At1g54445 protein interactome, revealing potential stress-responsive protein complexes. For spatial analysis, use immunohistochemistry with At1g54445 antibody on tissue sections from stressed plants to determine tissue-specific and cell-type-specific responses, correlating protein localization with physiological markers of stress response .

Advanced applications include chromatin immunoprecipitation (ChIP) if At1g54445 is suspected to associate with chromatin or regulate gene expression during stress. Design experiments using promoter-reporter constructs in conjunction with immunolabeling to correlate transcriptional activity with protein presence in specific cellular compartments . For systems biology approaches, integrate At1g54445 antibody-generated proteomics data with transcriptomics and metabolomics datasets to position this protein within broader stress response networks.

When designing these experiments, incorporate appropriate controls for antibody specificity under stress conditions, as protein modifications induced by stress might affect epitope recognition. This comprehensive approach utilizing At1g54445 antibody enables mechanistic insights into plant stress adaptation strategies and potentially identifies novel intervention points for improving crop resilience.

What methodological approaches are available for studying post-translational modifications of the At1g54445 protein?

Studying post-translational modifications (PTMs) of the At1g54445 protein requires specialized methodological approaches centered around the antibody. Begin with phospho-specific detection by developing or acquiring antibodies that specifically recognize phosphorylated residues of At1g54445. Alternatively, immunoprecipitate the protein using the standard At1g54445 antibody followed by phospho-specific staining or mass spectrometry analysis .

For comprehensive PTM profiling, implement the following workflow: First, immunoprecipitate At1g54445 protein from Arabidopsis tissues under different conditions using the antibody. Then perform mass spectrometry analysis with specific enrichment strategies for different modifications (phosphorylation, ubiquitination, SUMOylation, acetylation, etc.). Design parallel experiments using 2D gel electrophoresis followed by western blotting with At1g54445 antibody to visualize charge and mass shifts indicative of modifications .

To study dynamic PTM changes, design pulse-chase experiments combined with immunoprecipitation using At1g54445 antibody. For site-specific modification analysis, generate mutant Arabidopsis lines with alanine substitutions at predicted modification sites and compare immunoprecipitation results between wild-type and mutant proteins. Consider developing a panel of modification-specific antibodies for the most relevant PTMs identified through mass spectrometry .

Present PTM data in comprehensive tables showing modification types, modified residues, abundances under different conditions, and predicted functional impacts. This methodological approach not only identifies modifications but elucidates their biological significance in regulating At1g54445 protein function, stability, localization, and interaction partners, providing deeper insights into its role in plant biology.

How can computational approaches enhance the interpretation of At1g54445 antibody experimental data?

Computational approaches significantly enhance At1g54445 antibody data interpretation through multiple sophisticated methodologies. Begin by implementing machine learning algorithms for automated image analysis of immunohistochemistry or immunofluorescence data, which can detect subtle patterns in protein distribution and co-localization that might be missed by visual inspection .

Develop quantitative models integrating antibody-derived protein expression data with publicly available transcriptomic databases specific to Arabidopsis thaliana. This enables prediction of regulatory network interactions and identification of potential transcription factors or miRNAs controlling At1g54445 expression. For structural biology applications, use homology modeling and molecular dynamics simulations to predict the three-dimensional structure of the At1g54445 protein and map the epitope recognized by the antibody .

Implement Bayesian statistical frameworks for analyzing antibody-generated data with inherent variability, which can incorporate prior knowledge about protein behavior and experimental uncertainty. Design custom bioinformatics pipelines to integrate At1g54445 antibody data with other omics datasets using methods such as weighted gene correlation network analysis (WGCNA) or Gaussian graphical models .

For mechanistic insights, use systems biology approaches to position At1g54445 within broader functional networks by correlating antibody-detected expression patterns with phenotypic data across different experimental conditions. These computational strategies transform antibody-generated data from descriptive observations into predictive models of protein function, regulation, and interaction networks, significantly advancing understanding of At1g54445's biological roles in Arabidopsis thaliana.

What are the most common technical challenges when working with At1g54445 antibody and how can they be resolved?

When working with At1g54445 antibody, researchers commonly encounter several technical challenges that require systematic troubleshooting approaches. One frequent issue is high background signal in immunoblotting or immunostaining applications. To resolve this, implement more stringent blocking conditions (increasing BSA concentration from 3% to 5%, or adding 0.1-0.3% Triton X-100 to blocking buffer) and optimize antibody dilutions through careful titration experiments (testing ranges from 1:500 to 1:5000) .

Another common challenge is weak or inconsistent signal intensity. Address this by enhancing antigen retrieval methods for fixed tissues (testing multiple buffer systems at varying pH levels) or modifying protein extraction protocols to preserve epitope integrity (comparing RIPA, NP-40, and other lysis buffers). For samples with low target protein abundance, implement signal amplification strategies such as biotin-streptavidin systems or tyramide signal amplification .

Cross-reactivity with non-target proteins can be resolved through additional pre-adsorption steps or by increasing washing stringency. When At1g54445 antibody performs well in one application (e.g., western blotting) but poorly in another (e.g., immunoprecipitation), optimize buffer conditions specifically for each application, adjusting salt concentration, detergent types, and pH .

Create a detailed troubleshooting matrix documenting modifications to protocols and their outcomes across multiple experiments. This methodical approach not only resolves immediate technical issues but generates valuable protocol optimization data that enhances reproducibility and experimental success rates across different applications of the At1g54445 antibody.

How can I optimize immunoprecipitation protocols specifically for At1g54445 antibody?

Optimizing immunoprecipitation (IP) protocols for At1g54445 antibody requires systematic methodology refinement. Begin by comparing different lysis buffer compositions—test RIPA buffer against gentler alternatives like NP-40 or digitonin-based buffers that better preserve protein-protein interactions. For plant tissues specifically, incorporate plant-specific protease inhibitor cocktails containing inhibitors of papain-like enzymes common in Arabidopsis .

Systematically test antibody-to-lysate ratios to determine optimal binding conditions. Prepare a dilution series (typically 1-10 μg antibody per mg of total protein) and assess precipitation efficiency through western blotting of both precipitated material and supernatant. Compare different antibody immobilization methods, including direct conjugation to beads, protein A/G beads, or magnetic beads, to determine which provides optimal capture efficiency with minimal background .

Optimize incubation parameters through controlled experiments testing different temperatures (4°C vs. room temperature) and durations (1 hour vs. overnight). For washing steps, test buffers with increasing stringency (varying salt concentrations from 150mM to 500mM NaCl) while monitoring both target protein retention and contaminant removal .

Consider native versus crosslinked IP approaches—implement reversible crosslinking with formaldehyde or DSP (dithiobis(succinimidyl propionate)) to capture transient interactions, followed by controlled reversal before SDS-PAGE analysis. Document all optimization parameters in a comprehensive table, including quantitative measurements of precipitation efficiency and background levels for each condition. This methodological optimization creates a robust, reproducible IP protocol specifically tailored to At1g54445 antibody characteristics and the unique properties of plant tissue samples.

What are the methodological considerations for using At1g54445 antibody in plant tissue immunohistochemistry?

Implementing At1g54445 antibody in plant tissue immunohistochemistry requires specialized methodological considerations addressing the unique challenges of plant samples. First, optimize fixation protocols by comparing different fixatives (4% paraformaldehyde, Carnoy's solution, or glutaraldehyde-based mixtures) and duration times (2-24 hours) to preserve tissue architecture while maintaining epitope accessibility .

Develop effective tissue permeabilization strategies to overcome plant cell wall barriers without compromising antigen integrity. Test enzymatic digestion methods (using combinations of cellulase, hemicellulase, and pectinase) against physical permeabilization using detergents (Triton X-100, Tween-20) at various concentrations. For recalcitrant tissues, implement freeze-thaw cycles or moderate vacuum infiltration to enhance antibody penetration .

Address plant-specific challenges like autofluorescence by implementing quenching protocols (using sodium borohydride or Sudan Black B) or selecting detection systems with emission spectra distinct from chlorophyll and cell wall autofluorescence. Optimize antigen retrieval methods specifically for plant tissues, testing heat-induced epitope retrieval with citrate buffer against enzymatic retrieval methods .

Design appropriate controls including:

• Pre-immune serum controls at matching concentrations

• Absorption controls using recombinant At1g54445 protein

• Tissue sections from knockout or knockdown plant lines

Document tissue-specific optimization parameters in detailed protocols, including section thickness, pretreatment methods, antibody concentration, incubation times, and detection system specifications. These methodological refinements ensure specific localization of At1g54445 protein in plant tissues while minimizing artifacts common in plant immunohistochemistry, providing reliable spatial information about protein expression patterns across different cell types and developmental stages.