ASK7 Antibody

What is ASK7 and why is it important in plant research?

ASK7 (Arabidopsis SKP1-like 7) is a protein found in Arabidopsis thaliana that functions as part of the SCF (Skp1-Cullin-F-box) ubiquitin ligase complex, which plays critical roles in protein degradation pathways essential for plant development and environmental responses. The ASK7 protein participates in ubiquitin-mediated proteolysis, a fundamental cellular process that regulates numerous aspects of plant growth, development, and stress responses. Antibodies against ASK7 enable researchers to detect, quantify, and localize this protein in experimental systems, providing insights into plant molecular signaling networks .

What is the recommended dilution range for ASK7 antibody in Western blotting?

For Western blotting applications using ASK7 rabbit polyclonal antibody with Arabidopsis thaliana samples, the recommended dilution range is 1:50 to 1:200 . This relatively concentrated dilution range suggests the antibody may have moderate affinity or the target protein might be expressed at relatively low levels. Researchers should optimize the dilution for their specific experimental conditions by testing serial dilutions and considering factors such as protein concentration, detection method sensitivity, and incubation times.

What sample types can be used with ASK7 antibody?

The ASK7 rabbit polyclonal antibody has been validated for use with samples derived from Arabidopsis thaliana . Appropriate sample types include total protein extracts, subcellular fractions, immunoprecipitates, and potentially recombinant proteins from Arabidopsis expression systems. When preparing samples, researchers should consider using protease inhibitors to prevent degradation of the target protein and optimize extraction buffers to maintain protein integrity and native conformation.

How should ASK7 antibody be stored and handled for optimal performance?

For maintaining optimal activity, antibodies like the ASK7 rabbit polyclonal should be stored according to manufacturer recommendations. Generally, primary antibodies are stored at -20°C for long-term storage, with working aliquots kept at 4°C to minimize freeze-thaw cycles. Proper handling includes avoiding repeated freeze-thaw cycles, using sterile technique when preparing aliquots, and storing in appropriate buffers that may contain stabilizing proteins or preservatives. Short-term storage at 4°C is typically suitable for a few weeks, while long-term storage at -20°C or -80°C preserves activity for months to years.

How can cross-reactivity be assessed when working with ASK7 antibody?

When evaluating ASK7 antibody specificity, researchers should implement rigorous controls to assess potential cross-reactivity, particularly since polyclonal antibodies may recognize multiple epitopes. A comprehensive approach includes:

• Negative controls using samples from ASK7 knockout/knockdown lines

• Peptide competition assays where the antibody is pre-incubated with excess antigen

• Western blotting against recombinant ASK7 protein alongside total protein extracts

• Analysis of multiple plant tissues with varying ASK7 expression levels

What are the recommended protocols for optimizing Western blot detection of ASK7?

To optimize Western blot detection of ASK7 using the rabbit polyclonal antibody, researchers should consider implementing the following protocol refinements:

Similar to the methodical approach used in validating antibodies for specific detection of proteins like AR-V7 , researchers should systematically test these parameters to determine optimal conditions for ASK7 detection while monitoring signal-to-noise ratio throughout the optimization process.

How can immunoprecipitation with ASK7 antibody be optimized for protein interaction studies?

For protein interaction studies using ASK7 antibody in immunoprecipitation experiments, researchers should consider the following optimization strategies:

• Pre-clear lysates to reduce non-specific binding

• Optimize antibody concentration (typically 2-5 μg per mg of total protein)

• Test different bead types (Protein A/G, magnetic vs. agarose)

• Evaluate various washing stringencies to balance specific signal retention with background reduction

• Consider crosslinking the antibody to beads to prevent antibody co-elution

• Use appropriate negative controls (non-immune IgG, ASK7-knockout samples)

When analyzing results, particularly when investigating protein-protein interactions within the SCF complex, researchers should be mindful that interactions may be transient or condition-dependent, requiring stabilization approaches such as crosslinking or detergent optimization. Quantitative analysis of immunoprecipitation results can be enhanced by normalization to input samples and careful validation of interacting partners through reciprocal immunoprecipitation.

What considerations are important when using ASK7 antibody alongside other techniques for studying ubiquitin-mediated pathways?

When integrating ASK7 antibody detection with other methods to study ubiquitin-mediated pathways, researchers should consider:

• Complementary approaches to validate interactions (yeast two-hybrid, proximity ligation assays)

• Temporal considerations in experimental design to capture dynamic ubiquitination events

• Inhibitor treatments to stabilize otherwise transient interactions (proteasome inhibitors)

• Controls for ubiquitination status (ubiquitin antibodies, deubiquitinase inhibitors)

• Genetic approaches (mutants, overexpression lines) to validate antibody-based findings

This multi-technique approach resembles the comprehensive methodology described for simultaneous target discovery and antibody generation in research contexts , where integration of multiple experimental approaches provides more robust and reliable data than any single method alone.

How can researchers address contradictory results when using ASK7 antibody in different experimental conditions?

When confronting contradictory results using ASK7 antibody across different experimental conditions, consider implementing the following systematic troubleshooting approach:

• Antibody validation: Re-confirm antibody specificity using knockout/knockdown controls and recombinant protein standards

• Protocol standardization: Document and control all experimental variables (buffer composition, incubation times, temperatures)

• Sample preparation variations: Assess if differential extraction methods affect protein conformation or complex integrity

• Post-translational modifications: Investigate if modifications alter epitope recognition under different conditions

• Environmental influences: Evaluate if growth conditions, stress treatments, or developmental stages affect ASK7 expression or localization

• Technical replication: Increase biological and technical replicates to assess variability

• Alternative antibodies: When possible, utilize alternative antibodies targeting different epitopes of ASK7

Similar to the comparative antibody evaluation approach demonstrated in AR-V7 detection research , systematically evaluating each variable can help identify sources of contradictory results and determine whether they represent true biological phenomena or technical artifacts.

What controls should be included when using ASK7 antibody in immunoblotting experiments?

A comprehensive set of controls for ASK7 antibody immunoblotting should include:

• Positive control: Recombinant ASK7 protein or samples with confirmed ASK7 expression

• Negative control: Samples from ASK7 knockout/knockdown plants or tissues known not to express ASK7

• Loading control: Detection of a housekeeping protein (e.g., actin, tubulin) to normalize expression

• Antibody specificity control: Pre-incubation of antibody with immunizing peptide to block specific binding

• Secondary antibody control: Omitting primary antibody to assess non-specific binding of secondary antibody

• Molecular weight marker: To confirm detection at expected molecular weight

• Treatment controls: Appropriate vehicle controls for any treatments that may affect ASK7 expression

This systematic approach to controls is consistent with best practices in antibody validation demonstrated in publications examining antibody specificity and sensitivity , ensuring that experimental outcomes can be interpreted with confidence.

How can researchers quantitatively analyze ASK7 expression across different experimental conditions?

For quantitative analysis of ASK7 expression across experimental conditions, researchers should implement:

• Standardized protein quantification: Use reliable methods (BCA, Bradford) to ensure equal loading

• Normalization strategies: Express ASK7 signal relative to housekeeping proteins or total protein stains

• Linear detection range: Validate that signal intensity falls within the linear range of detection

• Technical replicates: Include multiple technical replicates for statistical robustness

• Image acquisition optimization: Use appropriate exposure settings to avoid saturation

• Quantification software: Utilize specialized software for densitometric analysis

• Statistical analysis: Apply appropriate statistical tests to determine significance of differences

This quantitative approach ensures rigorous analysis similar to methodologies employed in antibody characterization studies .

What advanced microscopy techniques can be coupled with ASK7 antibody for subcellular localization studies?

While the ASK7 antibody information primarily indicates Western blot applications , researchers interested in subcellular localization studies might consider adapting the antibody for immunofluorescence microscopy through careful validation. Advanced microscopy techniques potentially compatible with ASK7 antibody include:

• Confocal microscopy: For high-resolution imaging of ASK7 localization within plant cells

• Super-resolution microscopy (STED, STORM, PALM): For nanoscale resolution of ASK7 distribution

• Proximity ligation assay (PLA): For detecting ASK7 interactions with other proteins in situ

• Fluorescence resonance energy transfer (FRET): For studying ASK7 protein-protein interactions

• Correlative light and electron microscopy (CLEM): For correlating ASK7 localization with ultrastructural features

• Live-cell imaging: Using fluorescently tagged ASK7 to complement antibody-based fixed cell imaging

For each technique, researchers should:

• Validate antibody specificity in immunocytochemistry applications

• Optimize fixation and permeabilization conditions for plant cells

• Include appropriate controls (peptide competition, secondary-only, known localization markers)

• Consider dual labeling with organelle markers to confirm subcellular compartmentalization

These advanced imaging approaches provide spatial context to biochemical data, enhancing understanding of ASK7 function in plant cellular processes.

How can cutting-edge AI-based design approaches be applied to developing improved ASK7 antibodies?

Recent advances in AI-driven protein design, such as RFdiffusion, offer promising approaches for developing next-generation ASK7 antibodies with enhanced specificity and sensitivity. Implementation strategies include:

• Epitope optimization: Using AI algorithms to identify optimal ASK7 epitopes with high immunogenicity and specificity

• Antibody structure prediction: Employing models like RFdiffusion to design antibody complementarity-determining regions (CDRs) with improved binding characteristics

• Humanization for mammalian expression: Adapting plant-reactive antibodies for production in mammalian systems

• Affinity maturation in silico: Computational screening of antibody variants before experimental validation

• Format diversification: Designing single-chain variable fragments (scFvs) or nanobodies as alternatives to conventional antibodies

Similar to approaches described for antibody design using RFdiffusion , these AI-based methods could enhance ASK7 antibody performance by designing antibody loops—the flexible regions responsible for binding—with improved specificity and affinity. This would enable development of antibodies that can detect ASK7 with higher sensitivity, potentially expanding applications beyond Western blotting to include immunoprecipitation, ChIP-seq, or immunofluorescence.

How can researchers address weak or absent signals when using ASK7 antibody in Western blotting?

When encountering weak or absent signals with ASK7 antibody in Western blotting, researchers should systematically troubleshoot using this decision tree approach:

• Protein extraction and loading:

  • Increase protein concentration (50-100 μg total protein)

  • Verify protein transfer efficiency with reversible staining

  • Ensure appropriate sample preparation (fresh extraction, protease inhibitors)

• Antibody conditions:

  • Use higher antibody concentration (try 1:25 dilution)

  • Extend primary antibody incubation (overnight at 4°C)

  • Test fresh antibody aliquot to rule out degradation

• Detection sensitivity:

  • Switch to more sensitive detection system (enhanced chemiluminescence)

  • Increase exposure time during imaging

  • Consider signal amplification methods

• Biological considerations:

  • Verify ASK7 expression in selected tissue/condition

  • Consider treatments that might upregulate ASK7 expression

  • Evaluate developmental stage appropriateness

This systematic approach parallels the rigorous troubleshooting methodology applied in antibody validation studies for challenging targets , helping researchers isolate and address specific factors affecting detection.

What strategies can address non-specific binding when using ASK7 polyclonal antibody?

To reduce non-specific binding with ASK7 polyclonal antibody, researchers should implement these optimization strategies:

• Blocking optimization:

  • Test alternative blocking agents (5% BSA, 5% milk, commercial blockers)

  • Extend blocking time (2-3 hours at room temperature)

  • Add 0.1-0.3% Tween-20 to blocking solution

• Washing optimization:

  • Increase washing duration and frequency

  • Test different detergent concentrations in wash buffer

  • Consider alternative detergents (Triton X-100, NP-40)

• Antibody conditions:

  • Pre-absorb antibody with plant extract from unrelated species

  • Dilute antibody in blocking buffer with 0.1% Tween-20

  • Filter antibody solution before use

• Sample preparation:

  • Include additional clearing steps in sample preparation

  • Test alternative extraction buffers

  • Consider subcellular fractionation to enrich for target compartment

This approach aligns with strategies employed in antibody validation studies that emphasize the importance of optimizing conditions to achieve specific signal with minimal background .

How can researchers validate antibody specificity in ASK7 knockout or knockdown lines?

A rigorous approach to validating ASK7 antibody specificity using genetic controls involves:

• Genetic material preparation:

  • Obtain or generate ASK7 T-DNA insertion lines or CRISPR/Cas9 knockout lines

  • Develop RNAi or amiRNA knockdown lines with varying degrees of ASK7 reduction

  • Create complementation lines reintroducing ASK7 into knockout background

• Validation experimental design:

  • Extract protein from wild-type, knockout/knockdown, and complemented lines

  • Process all samples identically

  • Load equal amounts of total protein

  • Probe with ASK7 antibody and control antibodies

• Expected outcomes for specific antibody:

  • Strong signal in wild-type at expected molecular weight

  • Absent signal in knockout line

  • Reduced signal in knockdown lines proportional to knockdown efficiency

  • Restored signal in complementation line

• Quantitative analysis:

  • Perform densitometric analysis

  • Normalize to loading controls

  • Calculate signal reduction percentages

• Documentation:

  • Record complete methodological details

  • Include all validation data in publications

  • Note any unexpected bands or patterns

This validation approach follows principles similar to those employed in studies that carefully assessed antibody specificity using genetic controls and complementary techniques .

How can ASK7 antibody be integrated into phospho-proteomics workflows to study post-translational modifications?

Integrating ASK7 antibody into phospho-proteomics workflows enables investigation of post-translational modifications affecting SCF complex function. Implementation strategies include:

• Sequential immunoprecipitation workflow:

  • First IP: Enrich ASK7 using validated antibody

  • Elution: Gentle conditions to maintain PTMs

  • Second IP: Enrich with phospho-specific antibodies

  • MS analysis: Identify phosphorylation sites

• Phosphatase treatment controls:

  • Split samples for +/- phosphatase treatment

  • Compare ASK7 detection patterns

  • Identify mobility shifts indicative of phosphorylation

• 2D gel electrophoresis application:

  • Separate proteins by isoelectric point and molecular weight

  • Transfer and probe with ASK7 antibody

  • Identify different ASK7 isoforms by position

• Enrichment strategy integration:

  • Combine TiO₂ or IMAC phosphopeptide enrichment with ASK7 immunoprecipitation

  • Apply targeted mass spectrometry to identify ASK7 phosphopeptides

  • Quantify changes in phosphorylation under different conditions

This integrative approach draws on principles similar to those used in comprehensive protein characterization studies , providing deeper insights into ASK7 regulation through post-translational modifications.

What are the considerations for using ASK7 antibody in chromatin immunoprecipitation (ChIP) experiments?

While the ASK7 antibody is primarily validated for Western blotting , researchers interested in exploring potential chromatin associations through ChIP should consider:

• Antibody suitability assessment:

  • Verify antibody specificity in IP experiments before ChIP

  • Test fixation compatibility (formaldehyde crosslinking may mask epitopes)

  • Determine optimal antibody concentration for chromatin capture

• Experimental design considerations:

  • Include appropriate positive controls (known SCF-associated regions)

  • Use negative controls (transcriptionally inactive regions)

  • Include input normalization and IgG control immunoprecipitations

  • Consider spike-in normalization for quantitative comparisons

• Protocol optimization:

  • Test different crosslinking conditions (time, concentration)

  • Optimize sonication parameters for ideal fragment size

  • Evaluate different washing stringencies

  • Consider native ChIP alternatives if crosslinking reduces efficiency

• Validation approaches:

  • Confirm enrichment by qPCR before sequencing

  • Validate findings with orthogonal methods (e.g., reporter assays)

  • Consider sequential ChIP to identify co-occupancy with other factors

This methodical approach to applying antibodies in new experimental contexts draws on principles of careful validation and optimization described in antibody characterization literature .

How can ASK7 antibody be utilized in developing plant-specific biosensors or diagnostic tools?

Innovative applications of ASK7 antibody in biosensor development could include:

• Antibody-based biosensor platforms:

  • Surface plasmon resonance (SPR) sensors for real-time ASK7 detection

  • Quartz crystal microbalance (QCM) devices for quantitative measurements

  • Electrochemical impedance spectroscopy (EIS) sensors for field applications

  • Lateral flow immunoassays for rapid screening

• Engineering considerations:

  • Antibody immobilization strategies (oriented coupling via Fc region)

  • Surface chemistry optimization (self-assembled monolayers, polymer brushes)

  • Signal amplification methods (nanomaterials, enzymatic reactions)

  • Sample preparation for plant extracts

• Application scenarios:

  • Monitoring ASK7 expression as biomarker for plant stress responses

  • High-throughput screening of mutant collections

  • Environmental monitoring of plant responses to field conditions

  • Quality control in agricultural biotechnology

• Validation requirements:

  • Determine detection limits and dynamic range

  • Assess matrix effects from plant extracts

  • Establish reproducibility across biological replicates

  • Compare with established quantification methods

These innovative applications build on emerging technologies for antibody-based diagnostics and monitoring systems while adapting them to plant science applications, drawing inspiration from rapid methodologies developed for other research contexts .

How might AI-driven antibody design improve next-generation ASK7 antibodies?

Building on recent advances in AI-driven protein design like RFdiffusion , future ASK7 antibody development could benefit from:

• Enhanced epitope targeting:

  • Computational identification of highly specific ASK7 epitopes distinct from other ASK family proteins

  • Design of antibodies with reduced cross-reactivity to related proteins

  • Creation of conformation-specific antibodies for detecting functional states

• Performance optimization:

  • Fine-tuning binding affinity for different applications

  • Designing antibodies compatible with multiple techniques (WB, IP, IF, ChIP)

  • Engineering temperature and buffer stability for robust performance

• Novel formats development:

  • Single-domain antibodies for enhanced tissue penetration

  • Bi-specific antibodies to simultaneously detect ASK7 and interaction partners

  • Antibody fragments with improved recombinant production

• Validation approaches:

  • Rigorous testing against panels of ASK family proteins

  • Comparison with conventional antibodies using standardized assays

  • Structural validation of antibody-antigen complexes

Recent breakthroughs in using RFdiffusion to design human-like antibodies suggest that computational approaches could revolutionize the development of research antibodies with unprecedented specificity and versatility for challenging research applications.

What emerging methodologies might complement antibody-based detection of ASK7 in future research?

Emerging technologies that could complement traditional antibody-based ASK7 detection include:

• Proximity-based protein detection:

  • APEX2-based proximity labeling to identify ASK7 interaction networks

  • BioID approaches for mapping ASK7 protein neighborhoods

  • Split-protein complementation assays for studying dynamic interactions

• Advanced imaging techniques:

  • Expansion microscopy for enhanced resolution of ASK7 localization

  • Light-sheet microscopy for 3D visualization in intact tissues

  • Single-molecule tracking to monitor ASK7 dynamics in living cells

• Genetic tagging strategies:

  • CRISPR knock-in of epitope or fluorescent tags at endogenous loci

  • Nanobody-based detection of tagged ASK7 in living systems

  • Degron-based systems for studying ASK7 function through rapid depletion

• High-throughput phenotyping:

  • Automated image analysis of ASK7-dependent phenotypes

  • Single-cell transcriptomics correlated with ASK7 protein levels

  • Plant phenomics approaches for field-scale assessment of ASK7 function

These complementary approaches, drawing inspiration from rapid methodological developments in related fields , could provide multimodal insights into ASK7 biology beyond what antibody-based detection alone can achieve.