ASK21 Antibody

What is ASK21 and what is its role in plant biology?

ASK21 (SKP1-like protein 21) is a member of the 21-gene ASK family in Arabidopsis thaliana that encodes Skp1-like polypeptide subunits . These proteins function as components of SCF-type E3 ubiquitin ligase complexes, which target specific proteins for ubiquitination and subsequent degradation by the 26S proteasome. ASK proteins contribute to substrate specificity within SCF complexes and are involved in numerous developmental and environmental response pathways in plants.

The ASK21 protein specifically has been found to interact with certain F-box proteins, including potential involvement with FBW2, which targets AGO1 for degradation . Understanding ASK21's function helps researchers investigate plant protein degradation pathways and their regulation.

What validation methods should I use for ASK21 antibodies?

When validating ASK21 antibodies for research, follow these methodological approaches:

• Western blot validation: Run protein extracts from wild-type Arabidopsis tissues alongside ask21 mutant tissues (if available) or tissues where ASK21 is known to be differentially expressed.

• Specificity testing: Test cross-reactivity with other ASK family members, particularly those with high sequence similarity, using recombinant proteins.

• Application-specific validation: For each intended application (WB, ELISA, etc.), perform positive and negative controls.

• Multiple antibody approach: When possible, validate findings using multiple antibodies targeting different epitopes of ASK21 .

Remember that proper antibody validation is critical for experimental reproducibility. As noted in antibody validation literature, "after selecting an antibody, we recommend performing proper context-specific validation of the antibody for the research study in question" .

What are the optimal sample preparation methods for ASK21 detection?

For optimal ASK21 detection in plant tissues:

For Western blotting:

• Extract total proteins using a buffer containing 50mM Tris-HCl pH 7.5, 150mM NaCl, 1% Triton X-100, and protease inhibitor cocktail

• Include phosphatase inhibitors if phosphorylation status may be relevant

• Use fresh tissue when possible; if frozen, grind in liquid nitrogen before adding buffer

• Recommended protein amount: 20-40μg per lane

For immunoprecipitation:

• Use non-denaturing conditions to preserve protein-protein interactions

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Incubate with ASK21 antibody overnight at 4°C with gentle rotation

How does ASK21 expression vary across different tissues and developmental stages?

ASK21 expression patterns exhibit tissue specificity and developmental regulation. Based on quantitative RT-PCR analyses of the ASK gene family:

When designing experiments, consider these expression patterns to maximize detection probability. For optimal results, use tissues known to have higher ASK21 expression levels or consider using transgenic plants with ectopic expression if studying tissues with naturally low expression .

What controls should be included when using ASK21 antibodies in experimental work?

Include these critical controls in your experimental design:

• Positive control: Include samples known to express ASK21 (based on qRT-PCR data)

• Negative control:

  • If available, use ask21 knockout/knockdown plant tissues

  • Use pre-immune serum or isotype-matched control antibody

• Loading control: Include detection of a constitutively expressed protein

• Antibody specificity control: Test on recombinant ASK21 protein alongside other ASK family members

• Signal validation: Include a secondary antibody-only control

For immunohistochemistry, include peptide competition assays where the antibody is pre-incubated with excess immunizing peptide to verify signal specificity .

How can I optimize Western blot protocols for ASK21 detection?

For optimal Western blot detection of ASK21:

• Sample preparation:

  • Extract proteins in buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, and protease inhibitors

  • Heat samples at 95°C for 5 minutes in Laemmli buffer with DTT or β-mercaptoethanol

• Gel electrophoresis:

  • Use 12-15% SDS-PAGE gels

  • Load 20-40μg total protein per lane

  • Include molecular weight markers

• Transfer conditions:

  • Transfer to PVDF membrane (preferable over nitrocellulose for plant proteins)

  • Use wet transfer at 100V for 1 hour or 30V overnight at 4°C

• Blocking:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • Alternative: 3% BSA in TBST (if phosphorylation status matters)

• Antibody incubation:

  • Primary: Dilute ASK21 antibody 1:1000 in 1% milk/TBST, incubate overnight at 4°C

  • Secondary: Anti-rabbit HRP at 1:10,000 in 2% milk/TBST for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence reagent

  • Exposure time: Start with 30 seconds, adjust as needed

How can ASK21 antibodies be used to investigate protein-protein interactions in SCF complexes?

To investigate ASK21's role in SCF complexes:

• Co-immunoprecipitation (Co-IP):

  • Lyse plant tissues in non-denaturing buffer

  • Immunoprecipitate using ASK21 antibody

  • Analyze precipitates for SCF components (Cullin1, RBX1, F-box proteins)

  • The reciprocal approach can also be informative: IP with antibodies against known SCF components and probe for ASK21

• Bimolecular Fluorescence Complementation (BiFC):

  • Generate constructs expressing ASK21 fused to fragments of fluorescent proteins

  • Co-express with potential interaction partners similarly tagged

  • Visualize interactions by confocal microscopy

  • This approach has been successfully used for other ASK family members

• Yeast Two-Hybrid (Y2H):

  • Use ASK21 as bait to screen for interacting F-box proteins

  • Confirm interactions detected in Y2H using in planta methods

Research has shown that ASK proteins interact with F-box proteins like FBW2, which targets AGO1 for degradation . Similar approaches can be used to identify additional ASK21-specific interaction partners.

What are the challenges in distinguishing ASK21 function from other ASK family members?

Researchers face several challenges when studying ASK21 specifically:

• Sequence similarity:

  • The ASK gene family contains 21 members with varying degrees of similarity

  • ASK21 may share structural features with other family members

  • Antibody cross-reactivity must be rigorously tested

• Functional redundancy:

  • Multiple ASK proteins may perform similar functions

  • Single mutants may not display obvious phenotypes

  • Consider generating higher-order mutants or using inducible RNAi

• Methodological approach:

  • Combined transcriptomics, proteomics, and genetic approaches may be necessary

  • Use CRISPR/Cas9 to generate precise mutations or epitope tags

  • Consider tissue-specific or condition-specific analyses

• Data interpretation:

  • When phenotypes are observed, confirm ASK21 specificity through complementation

  • Use phylogenetic analysis to identify closest homologs for potential redundancy

To address these challenges, researchers can employ precise genetic approaches combined with specific antibody detection methods.

How can I use ASK21 antibodies in studying plant hormone signaling pathways?

ASK proteins, including ASK21, are involved in hormone signaling through their role in SCF complexes. To study ASK21's role:

• Hormone treatment experiments:

  • Treat plants with hormones (auxin, jasmonate, gibberellin, etc.)

  • Monitor ASK21 protein levels, subcellular localization, and complex formation

  • Compare with known hormone-responsive ASK proteins

• Genetic interaction studies:

  • Cross ask21 mutants with hormone signaling mutants

  • Analyze double mutant phenotypes for enhancement or suppression

  • Consider higher-order mutants with other ASK genes

• Immunohistochemistry in hormone-treated tissues:

  • Use ASK21 antibodies to visualize protein localization changes

  • Compare with hormone reporter lines

  • Quantify signal intensity changes

• Proteomics approach:

  • Perform immunoprecipitation of ASK21 before and after hormone treatments

  • Identify differential interaction partners by mass spectrometry

  • Validate key interactions with targeted approaches

Given that SCF complexes regulate multiple hormone pathways, ASK21 may have hormone-specific or condition-specific functions that can be revealed through these approaches .

What methods are available for studying post-translational modifications of ASK21?

To investigate post-translational modifications (PTMs) of ASK21:

• Phosphorylation analysis:

  • Immunoprecipitate ASK21 and analyze by phospho-specific staining

  • Use phosphatase treatments as controls

  • Consider phospho-proteomics approaches to identify specific sites

  • Generate phospho-specific antibodies for key sites

• Ubiquitination detection:

  • Co-IP ASK21 and probe for ubiquitin

  • Use proteasome inhibitors (MG132) to stabilize ubiquitinated forms

  • Consider ubiquitin remnant profiling by mass spectrometry

• Other PTMs:

  • SUMOylation can be detected by SUMO-specific antibodies

  • Acetylation can be analyzed by acetylation-specific antibodies

  • Mass spectrometry can identify unexpected modifications

• Functional consequences:

  • Correlate PTM status with SCF complex formation ability

  • Generate site-specific mutants to test functional importance

  • Monitor PTM changes during development or stress responses

These approaches can reveal regulatory mechanisms controlling ASK21 function in different contexts.

What steps should I take if my ASK21 antibody produces unexpected results?

When facing unexpected results with ASK21 antibodies, follow this systematic approach:

• Antibody validation:

  • Confirm antibody specificity with recombinant ASK21 protein

  • Test for cross-reactivity with other ASK family members

  • Consider epitope mapping to understand antibody binding sites

• Experimental conditions:

  • Adjust antibody concentration (try dilutions from 1:500 to 1:5000)

  • Modify blocking conditions (try BSA instead of milk)

  • Vary incubation times and temperatures

  • Test different detection methods

• Sample preparation:

  • Ensure proper tissue preparation and protein extraction

  • Check for proteolytic degradation (add additional protease inhibitors)

  • Consider subcellular fractionation if localization is an issue

• Controls and comparisons:

  • Include positive and negative controls in every experiment

  • Compare results with published data on ASK21 or related proteins

  • Consider using an alternative antibody targeting a different epitope

Thorough documentation of all optimization steps will help identify the source of unexpected results.

How can I verify that my ASK21 antibody is detecting the correct protein in complex plant samples?

To confirm antibody specificity in complex samples:

• Genetic verification:

  • Compare wild-type samples with ask21 mutants

  • Use overexpression lines as positive controls

  • Consider inducible systems for controlled expression

• Molecular verification:

  • Perform immunoprecipitation followed by mass spectrometry

  • Compare detected peptides with ASK21 sequence

  • Look for co-precipitating known interaction partners

• Immunodepleting experiments:

  • Pre-absorb antibody with recombinant ASK21

  • Use this depleted antibody as a control

  • Signal should be significantly reduced with depleted antibody

• Epitope competition:

  • Pre-incubate antibody with immunizing peptide

  • This should block specific binding in applications

  • Compare signal with and without peptide competition

These approaches provide multiple lines of evidence for antibody specificity.

How can ASK21 antibodies contribute to understanding plant stress response mechanisms?

ASK21 antibodies can advance plant stress research through:

• Stress-responsive SCF complex formation:

  • Monitor ASK21 protein levels during various stresses

  • Identify stress-specific interaction partners

  • Investigate changes in subcellular localization

• Comparative studies:

  • Compare ASK21 with other stress-responsive ASK proteins

  • Investigate potential redundancy or specialization

  • Correlate protein levels with transcriptomic data

• Post-translational modification research:

  • Analyze stress-induced PTMs of ASK21

  • Correlate modifications with altered interactions

  • Investigate signaling cascades leading to modification

• Translation to crop species:

  • Use information from Arabidopsis to identify ASK21 orthologs in crops

  • Develop antibodies for crop homologs

  • Investigate conservation of stress response mechanisms

By designing experiments that combine genetic approaches with protein-level analysis using ASK21 antibodies, researchers can gain deeper insights into plant stress adaptations.

What emerging technologies could enhance ASK21 antibody-based research?

Several cutting-edge approaches can be integrated with ASK21 antibody research:

• Proximity labeling proteomics:

  • Generate ASK21 fused to BioID or TurboID

  • Identify proteins in close proximity to ASK21 in vivo

  • Compare interactomes under different conditions

• Super-resolution microscopy:

  • Visualize ASK21 subcellular localization at nanometer resolution

  • Study co-localization with potential partners

  • Combine with FRET or FLIM for interaction studies

• Single-cell proteomics:

  • Analyze ASK21 levels in specific cell types

  • Correlate with single-cell transcriptomics

  • Understand cell-type specific functions

• CRISPR-based approaches:

  • Generate endogenously tagged ASK21 for antibody-independent detection

  • Create precise mutations in functional domains

  • Develop conditional degradation systems

• Deep learning antibody design:

  • Apply AI approaches to design more specific ASK21 antibodies

  • Optimize epitope selection based on structural predictions

  • Similar to approaches used for human antibody libraries

Integration of these technologies with traditional antibody-based approaches will provide more comprehensive understanding of ASK21 function.