ASK19 Antibody

What is ASK19 and what biological functions does it serve in plant systems?

ASK19 (Arabidopsis SKP1-like 19) is a plant protein that functions as a critical component of the SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase complex in Arabidopsis thaliana. The protein plays an essential role in ubiquitination pathways, which mark target proteins for proteasomal degradation. ASK19 belongs to the ASK family of proteins (Arabidopsis SKP1-like), which contains multiple members with potentially overlapping functions in protein degradation mechanisms. The protein is identified by Uniprot accession number O81058 and is specifically expressed in Arabidopsis thaliana tissues .

To study ASK19, researchers typically use techniques including Western blotting, immunoprecipitation, and immunohistochemistry with specific antibodies. The antibody against ASK19 allows visualization of protein localization, quantification of expression levels, and investigation of protein-protein interactions within the ubiquitination pathway. For optimal results, researchers should use positive controls (such as recombinant ASK19 protein) and negative controls (tissues from knockout mutants) to validate antibody specificity.

How should researchers validate ASK19 antibody specificity before experimental application?

Validating antibody specificity is crucial for generating reliable experimental data when studying ASK19. A comprehensive validation protocol should include multiple approaches to ensure the antibody recognizes only the target protein. Begin with Western blot analysis using wild-type Arabidopsis tissue extracts compared against ask19 knockout mutants. A specific antibody will show a band at the expected molecular weight (approximately 19-21 kDa for ASK19) in wild-type samples that is absent in knockout tissues.

For further validation, perform peptide competition assays where the antibody is pre-incubated with excess synthetic ASK19-specific peptide before immunoblotting. If the antibody is specific, this pre-incubation should eliminate or significantly reduce signal detection. Cross-reactivity testing against other ASK family members, particularly those with high sequence homology to ASK19, is essential given the multiple ASK proteins (ASK1-19) in Arabidopsis .

Immunoprecipitation followed by mass spectrometry can provide additional validation by confirming that the antibody pulls down ASK19 rather than other proteins. Finally, immunolocalization patterns should be compared with known subcellular distribution patterns of ASK19 and validated using fluorescently-tagged ASK19 expressed in plant cells. This multi-faceted approach ensures that experimental results obtained with the antibody accurately reflect ASK19 biology.

What are the optimal storage and handling conditions for maintaining ASK19 antibody activity?

Maintaining antibody activity through proper storage and handling is essential for consistent experimental results. ASK19 antibodies, like most research antibodies, should be stored at -20°C for long-term preservation and at 4°C for solutions in current use . Avoid repeated freeze-thaw cycles by aliquoting the antibody into single-use volumes upon receipt. Each freeze-thaw cycle can reduce antibody activity by 10-15%, potentially compromising experimental reproducibility.

For working solutions, dilute the antibody in appropriate buffer (typically PBS with 0.05% sodium azide) and add a carrier protein like BSA (0.1-1%) to prevent adhesion to storage tubes. Document the date of reconstitution and track the number of freeze-thaw cycles for each aliquot. When handling the antibody, use low-protein binding tubes and avoid vortexing, which can cause protein denaturation – instead, mix by gentle inversion or flicking.

Prior to each experimental application, centrifuge the antibody solution briefly (10,000 g for 5 minutes) to remove any aggregates that may have formed during storage. For critical experiments, perform a small-scale validation test with each new lot of antibody to ensure consistent performance compared to previously validated lots. Implementing these practices will maximize antibody shelf-life and ensure reliable detection of ASK19 protein in your experiments.

How can ASK19 antibody be used to investigate protein-protein interactions within the SCF complex?

Investigating protein-protein interactions involving ASK19 within the SCF complex requires sophisticated immunological approaches. Co-immunoprecipitation (Co-IP) represents the gold standard method, where ASK19 antibody is used to pull down the entire protein complex from plant tissue lysates. Begin by crosslinking the antibody to protein A/G beads to prevent antibody co-elution with the target proteins. After immunoprecipitation with ASK19 antibody, analyze the precipitated complexes by Western blotting with antibodies against suspected interaction partners (like cullin proteins, F-box proteins, or substrate proteins).

For more sensitive detection of transient or weak interactions, proximity ligation assays (PLA) can be employed, where ASK19 antibody is used alongside antibodies against potential interaction partners. A positive PLA signal, visualized as fluorescent dots, confirms that proteins are within 40 nm of each other in situ. Alternatively, bimolecular fluorescence complementation (BiFC) combined with immunofluorescence using ASK19 antibody can provide complementary evidence for protein interactions.

To determine if interactions are direct or mediated by other proteins, perform in vitro binding assays with purified recombinant proteins followed by detection with ASK19 antibody. Quantitative analysis of interaction dynamics can be achieved through fluorescence resonance energy transfer (FRET) by labeling ASK19 antibody with a donor fluorophore and the interaction partner's antibody with an acceptor fluorophore. These advanced applications provide comprehensive characterization of ASK19's role in SCF complex assembly and function in plant ubiquitination pathways.

What are the key methodological considerations when using ASK19 antibody for studying protein degradation pathways?

When investigating protein degradation pathways involving ASK19, several methodological considerations are critical for generating reliable data. First, design time-course experiments with proteasome inhibitors (like MG132) to capture the dynamic nature of ubiquitination and subsequent degradation processes. This approach allows visualization of substrate accumulation when proteasomal degradation is blocked, providing evidence for ASK19's involvement in specific degradation pathways.

Cell fractionation followed by immunoblotting with ASK19 antibody can reveal the subcellular compartments where ASK19-mediated ubiquitination occurs. This is important because different pools of the same protein may be regulated differently depending on their subcellular location. When performing ubiquitination assays, include appropriate controls such as deubiquitinase inhibitors (like PR-619) to prevent removal of ubiquitin chains during sample processing.

For studying substrate specificity, combine ASK19 antibody detection with techniques like tandem ubiquitin binding entity (TUBE) pulldowns to enrich for ubiquitinated proteins, followed by mass spectrometry to identify potential substrates. Validation of putative substrates should include in vivo co-immunoprecipitation with ASK19 antibody and in vitro reconstitution of ubiquitination using purified components.

Finally, when interpreting results, consider the potential redundancy among ASK family members (ASK1-19), as functional compensation might occur in single mutant backgrounds . Using ASK19 antibody in combination with genetic approaches (like multiple mutant combinations) provides the most comprehensive understanding of ASK19's specific contribution to protein degradation pathways.

How does the function of ASK19 differ from other ASK family proteins, and how can researchers experimentally distinguish them?

The ASK family in Arabidopsis thaliana comprises 19 members (ASK1-19) with varying degrees of sequence similarity and potentially overlapping functions . Distinguishing the specific function of ASK19 from other family members requires a combination of genetic, biochemical, and immunological approaches. At the protein level, ASK19 shares approximately 35-75% sequence identity with other ASK proteins, making specific antibody detection challenging but essential.

To experimentally distinguish ASK19 from other family members, researchers should first conduct epitope mapping to identify unique regions of ASK19 suitable for raising specific antibodies. Conduct Western blot analysis with the ASK19 antibody against recombinant proteins of all ASK family members to confirm specificity. Any cross-reactivity should be documented and considered when interpreting experimental results.

Functionally, ASK19 may participate in distinct SCF complexes by interacting with specific F-box proteins. Perform immunoprecipitation with ASK19 antibody followed by mass spectrometry to identify unique interaction partners not shared with other ASK proteins. Complementary approaches include yeast two-hybrid screening and in vitro binding assays with purified components.

Genetic approaches provide additional differentiation by analyzing phenotypes of single and higher-order mutants. Create transgenic Arabidopsis lines expressing epitope-tagged versions of ASK19 in ask19 mutant backgrounds for rescue experiments, then use both ASK19 antibody and epitope tag antibodies to verify expression patterns. Tissue-specific and developmental expression analysis using ASK19 antibody in immunohistochemistry can further distinguish its functional domain from other ASK proteins. This multi-faceted approach will reveal the unique biological roles of ASK19 within the broader context of SCF-mediated protein degradation.

What controls should researchers include when performing Western blot analysis with ASK19 antibody?

Rigorous Western blot analysis with ASK19 antibody requires a comprehensive set of controls to ensure data reliability and interpretability. As a positive control, include recombinant ASK19 protein at a known concentration to verify antibody recognition and establish a reference signal intensity. The negative control should consist of protein extracts from ask19 knockout mutants to confirm signal specificity.

To assess potential cross-reactivity with other ASK family members, perform parallel blots with extracts from plants overexpressing various ASK proteins. Include loading controls appropriate for plant samples, such as anti-actin or anti-GAPDH antibodies, to normalize protein loading across lanes. For developmental or stress-response studies, include reference samples from well-characterized conditions to provide context for experimental variations.

When evaluating post-translational modifications, include samples treated with relevant enzymes (phosphatases, deubiquitinases) as controls. For antibody validation, perform peptide competition assays where the primary antibody is pre-incubated with the immunizing peptide before membrane application – this should abolish specific signal while non-specific binding remains.

How can researchers optimize immunohistochemistry protocols when using ASK19 antibody in plant tissues?

Optimizing immunohistochemistry protocols for ASK19 detection in plant tissues requires addressing several plant-specific challenges. Begin with proper tissue fixation – test both cross-linking (4% paraformaldehyde) and precipitating (ethanol/acetic acid) fixatives to determine which better preserves ASK19 antigenicity while maintaining tissue morphology. Plant tissues often require longer fixation times (12-24 hours) compared to animal tissues due to cell wall barriers.

Antigen retrieval is critical when working with fixed plant tissues. Compare heat-induced epitope retrieval (citrate buffer, pH 6.0, 95°C for 10-20 minutes) with enzymatic methods (using cell wall degrading enzymes like driselase or pectinase) to optimize ASK19 epitope accessibility. Cell wall permeabilization requires additional steps beyond membrane permeabilization – incorporate a cocktail of cell wall degrading enzymes during the permeabilization step.

When blocking, use a combination of BSA (3-5%) with normal serum from the secondary antibody species, supplemented with 0.1-0.3% Triton X-100 to enhance penetration through plant cell walls. For primary antibody incubation with ASK19 antibody, extend incubation times (overnight to 48 hours at 4°C) and test a broader concentration range (1:50 to 1:1000) compared to animal tissues.

To reduce plant tissue autofluorescence, pretreat sections with sodium borohydride (0.1% for 10 minutes) or incorporate Sudan Black B (0.1% in 70% ethanol) after secondary antibody incubation. Include controls consisting of wild-type versus ask19 mutant tissues processed identically to validate signal specificity. Finally, optimize mounting media containing anti-fading agents compatible with plant tissue sections to prevent signal quenching during imaging. This plant-specific approach will maximize ASK19 detection sensitivity while minimizing background issues common in plant immunohistochemistry.

What are common troubleshooting strategies for inconsistent ASK19 antibody performance in experimental applications?

When facing inconsistent ASK19 antibody performance, systematic troubleshooting is essential for resolving technical issues. First, evaluate antibody quality by performing dot blots with recombinant ASK19 protein at various dilutions to determine if the antibody still recognizes its target. Antibody degradation often manifests as reduced sensitivity rather than complete signal loss.

For weak or absent signals in Western blotting, optimize protein extraction by testing multiple buffer compositions with different detergents (RIPA, NP-40, Triton X-100) and protease inhibitor cocktails specifically formulated for plant tissues. Plant proteases are particularly active and may degrade ASK19 during extraction. Additionally, test multiple antigen retrieval methods, as plant proteins can form complexes with phenolic compounds that mask epitopes.

If background is the primary issue, implement more stringent washing procedures with higher salt concentrations (up to 500 mM NaCl) in wash buffers to disrupt non-specific interactions. For plant tissues with high phenolic content, add polyvinylpyrrolidone (PVP) to extraction and washing buffers to sequester these compounds that can bind non-specifically to antibodies.

Batch-to-batch variability in antibody performance can be addressed by maintaining reference samples from successful experiments to benchmark new antibody lots. Create a detailed protocol log documenting exact conditions from successful experiments, including buffer compositions, incubation times, and temperatures.

For immunoprecipitation issues, test alternative lysis conditions and pre-clear lysates more extensively to remove components that cause non-specific binding. Cross-linking antibodies to beads can prevent heavy chain interference in subsequent immunoblotting. If experiments remain inconsistent despite these measures, consider epitope-tagging approaches (HA, FLAG, etc.) as an alternative strategy to study ASK19, using well-characterized commercial antibodies against these tags.

How should researchers interpret conflicting results between ASK19 protein levels detected by antibody versus mRNA expression data?

Discrepancies between ASK19 protein levels and mRNA expression data are common and biologically meaningful rather than experimental artifacts. When encountering such discrepancies, consider post-transcriptional and post-translational regulatory mechanisms. ASK19, as a component of the SCF complex involved in ubiquitination, may itself be subject to regulation through protein degradation pathways, creating a disconnect between mRNA and protein levels.

To systematically investigate these differences, perform time-course experiments measuring both mRNA (by qRT-PCR) and protein levels (by Western blot with ASK19 antibody) following relevant treatments or developmental stages. Calculate protein-to-mRNA ratios to identify conditions where post-transcriptional regulation may be occurring. Test whether protein synthesis inhibitors (cycloheximide) or proteasome inhibitors (MG132) affect ASK19 protein levels independently of mRNA changes.

For quantitative analysis, implement pulse-chase experiments with metabolic labeling followed by immunoprecipitation with ASK19 antibody to measure protein half-life under different conditions. This approach can reveal changes in protein stability that explain discrepancies with mRNA levels. Additionally, examine ASK19 for post-translational modifications using phospho-specific antibodies or mass spectrometry after immunoprecipitation, as these modifications often affect protein stability independent of transcription.

When publishing results showing such discrepancies, present both protein and mRNA data together with appropriate statistical analysis and clearly state the biological implications of the observed differences. This comprehensive approach transforms an apparent experimental contradiction into a valuable insight into the regulation of ASK19 function in plant biology.

What statistical approaches are recommended for quantifying ASK19 protein levels in comparative studies?

Robust statistical analysis of ASK19 protein quantification requires approaches that address both technical and biological variation. Begin with proper experimental design, including biological replicates (different plants/samples) and technical replicates (multiple measurements of the same sample) to distinguish these sources of variation. For Western blot quantification of ASK19, implement a nested ANOVA design that accounts for this hierarchical structure of variation.

When quantifying immunoblot band intensities, establish a standard curve using recombinant ASK19 protein to ensure measurements fall within the linear range of detection. For normalization, the choice of reference protein is critical – validate that traditional housekeeping proteins (actin, GAPDH, tubulin) maintain stable expression under your experimental conditions by testing multiple reference proteins and selecting the most consistent one.

For immunohistochemistry quantification, conduct cell-by-cell analysis rather than whole-tissue averages to capture population heterogeneity in ASK19 expression. Apply appropriate transformations (log, square root) to immunofluorescence intensity data that typically follow non-normal distributions before parametric statistical testing.

When comparing multiple experimental groups, avoid multiple t-tests, which inflate Type I error rates. Instead, use ANOVA followed by appropriate post-hoc tests with correction for multiple comparisons (Tukey's HSD, Bonferroni, or Benjamini-Hochberg procedures). For time-course experiments, repeated measures ANOVA or mixed-effects models better account for within-subject correlation.

For more complex experimental designs, consider multivariate approaches like principal component analysis (PCA) or partial least squares regression to identify patterns of ASK19 expression in relation to other measured variables. Present results with appropriate visualization methods (box plots with individual data points, not just bar graphs) and always report both effect sizes and p-values to communicate biological significance alongside statistical significance.

How can researchers effectively integrate ASK19 antibody-based protein data with other omics datasets?

Integrating ASK19 protein data with other omics datasets requires thoughtful data processing and statistical approaches to account for the different technical characteristics of each data type. Begin by establishing a common experimental framework where samples for different omics analyses (proteomics, transcriptomics, metabolomics) are derived from the same biological material whenever possible to minimize variation.

For integration with transcriptomics data, normalize ASK19 protein quantities detected by Western blotting or immunohistochemistry to facilitate comparison with normalized RNA-seq or microarray data. Apply correlation analyses (Pearson or Spearman depending on data distribution) between ASK19 protein levels and mRNA expression of genes involved in related biological processes. Network analysis approaches like weighted gene correlation network analysis (WGCNA) can identify modules of co-expressed genes that correlate with ASK19 protein levels across conditions.

When integrating with global proteomics data, use ASK19 antibody for targeted validation of mass spectrometry-based observations. Immunoprecipitation with ASK19 antibody followed by mass spectrometry (IP-MS) provides a powerful approach to identify the "interactome" of ASK19, which can then be mapped onto broader proteomics datasets to identify functional modules.

For multi-omics integration, implement advanced computational approaches such as Partial Least Squares (PLS) regression or Canonical Correlation Analysis (CCA) to identify relationships between ASK19 protein levels and patterns in other omics data. Pathway enrichment analyses incorporating ASK19 protein data with transcriptomics and metabolomics can reveal biological processes affected by ASK19 function.

Visualization is crucial for effective data integration – use heatmaps, network diagrams, and multi-layered visualizations that simultaneously display ASK19 protein levels alongside other omics data. Finally, validate key findings from integrated analyses using targeted experimental approaches, such as genetic perturbation of ASK19 followed by measurement of predicted effects on other molecular components. This comprehensive approach transforms individual datasets into a systems-level understanding of ASK19 biology.

What are the latest methodological advances in studying ASK19 protein dynamics in living plant cells?

Recent methodological advances have revolutionized the study of ASK19 protein dynamics in living plant cells, moving beyond static antibody-based detection to real-time visualization and quantification. Fluorescent protein fusions (ASK19-GFP, ASK19-mCherry) expressed under native promoters in ask19 mutant backgrounds provide minimally invasive systems for tracking protein movement and interactions. These constructs can be validated using ASK19 antibody to confirm that the fusion protein exhibits similar localization and functional properties as the endogenous protein.

Advanced microscopy techniques including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss in Photobleaching (FLIP) now allow measurement of ASK19 protein mobility and residence time in different subcellular compartments, providing insights into its dynamic association with the SCF complex. For studying protein-protein interactions in vivo, Förster Resonance Energy Transfer (FRET) and Bimolecular Fluorescence Complementation (BiFC) provide spatial information about ASK19 interactions that complements traditional co-immunoprecipitation approaches.

Optogenetic tools represent the cutting edge for studying ASK19 function, allowing light-controlled activation or inhibition of protein activity. Systems like light-inducible dimerization can trigger ASK19 association with specific partners on demand, enabling precise temporal control over complex formation. These approaches can be combined with fast-folding fluorescent reporters to visualize downstream effects of ASK19 activation.

For quantitative analysis of protein turnover, tandem fluorescent protein timers (tFTs) consisting of two fluorescent proteins with different maturation rates fused to ASK19 provide a ratiometric readout of protein age distributions within cells. This approach reveals heterogeneity in ASK19 stability across different cellular compartments or developmental stages without requiring antibody detection. These emerging technologies collectively provide unprecedented insights into the spatiotemporal regulation of ASK19 function in plant ubiquitination pathways.

How can comparative analysis between ASK family members inform our understanding of SCF complex functional diversity?

Comparative analysis across the ASK family provides critical insights into functional specialization and redundancy within SCF complexes. With 19 ASK proteins in Arabidopsis (ASK1-19), systematic approaches are needed to disentangle their specific roles . Begin with phylogenetic analysis to cluster ASK proteins based on sequence similarity, which often correlates with functional similarity. Using ASK19 antibody alongside antibodies against other ASK proteins (or epitope-tagged versions), compare expression patterns across tissues, developmental stages, and in response to environmental stimuli.

Immunoprecipitation with antibodies against different ASK proteins followed by mass spectrometry enables comparison of their interaction partners, particularly F-box proteins that confer substrate specificity to SCF complexes. This "interactome mapping" approach reveals both unique and overlapping functions. Complementary yeast two-hybrid or in vitro binding assays with purified components can determine binding affinities, providing quantitative insights into preferential interactions.

Genetic analysis comparing single and higher-order ask mutants reveals functional redundancy through enhancement of phenotypic severity in multiple mutants. For mechanistic understanding, in vitro reconstitution of SCF complexes with different ASK proteins allows comparison of ubiquitination activity toward various substrates. These biochemical assays can determine if differences in activity result from altered complex assembly, substrate recognition, or catalytic efficiency.

Advanced structural biology approaches, including cryo-electron microscopy of reconstituted SCF complexes containing different ASK proteins, reveal how subtle structural variations influence complex architecture and function. Finally, evolutionary analysis comparing ASK family composition across plant species provides insights into the emergence of functional specialization through gene duplication and divergence. This multi-faceted comparative approach transforms our understanding of the SCF complex from a single entity to a diverse family of ligases with specialized functions in plant development and environmental responses.

What emerging technologies are enhancing the capabilities of antibody-based detection for low-abundance proteins like ASK19?

Emerging technologies are dramatically improving detection sensitivity for low-abundance proteins like ASK19, enabling research previously hindered by detection limits. Signal amplification methods represent a major advance, with technologies like Tyramide Signal Amplification (TSA) enhancing immunohistochemical detection by generating multiple fluorophore deposits at antibody binding sites, increasing sensitivity up to 100-fold over conventional methods. This approach is particularly valuable for detecting ASK19 in tissues where it may be expressed at low levels.

Proximity ligation assays (PLA) have revolutionized protein interaction studies by generating a fluorescent signal only when two antibodies (e.g., against ASK19 and a potential interaction partner) bind within 40 nm of each other. This technology not only confirms protein proximity but amplifies the signal through rolling circle DNA amplification, enabling detection of even rare interaction events that would be missed by traditional co-immunoprecipitation.

Single-molecule detection techniques, including stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM), overcome the diffraction limit of conventional microscopy, allowing visualization of individual ASK19 molecules within cells when labeled with photoswitchable fluorophores conjugated to antibodies. These super-resolution approaches reveal spatial organization of ASK19 relative to other SCF components with nanometer precision.

For quantitative applications, digital ELISA platforms (like Simoa) use antibody-coated microbeads and single-molecule array technology to detect proteins at femtomolar concentrations, representing a 1000-fold improvement over traditional ELISA. This enables quantification of ASK19 from minimal sample volumes or from tissues where expression is extremely low.

Mass cytometry (CyTOF) combines flow cytometry with mass spectrometry by using antibodies labeled with rare earth metals instead of fluorophores, eliminating spectral overlap issues and allowing simultaneous detection of dozens of proteins, including ASK19, at the single-cell level. These technological advances collectively empower researchers to study ASK19 biology with unprecedented sensitivity and molecular resolution, opening new avenues for understanding its role in plant ubiquitination pathways.

ASK19 Antibody Validation Parameters: Performance Across Multiple Experimental Applications

Comparative Analysis of ASK Family Members: Structural and Functional Characteristics

Optimization Parameters for ASK19 Protein Extraction from Different Plant Tissues