ASK13 Antibody

What is ASK13 and why is it important in plant research?

ASK13 (Arabidopsis SKP1-like protein13; At3g60010) is a member of the SKP1 family of proteins found in Arabidopsis thaliana. It functions as a critical component in various cellular processes, particularly in seed germination and seedling growth under abiotic stress conditions. ASK13 is differentially regulated in different organs during seed development and germination, and is notably up-regulated during abiotic stress response .

The significance of ASK13 in plant research lies in its role in stress adaptation mechanisms. Understanding ASK13 function provides insights into how plants respond to environmental challenges, which has implications for developing stress-resistant crop varieties. Additionally, ASK13 interacts with various F-box proteins and other proteins independent of SCF (Skp, Cullin, F-box containing) complexes, suggesting involvement in multiple cellular pathways beyond protein degradation .

What types of antibodies are available for ASK13 detection?

While the search results don't specifically detail commercial antibodies targeting ASK13, researchers commonly use custom antibodies developed against specific epitopes of the ASK13 protein. For experimental purposes, researchers often create fusion proteins such as GFP-fused ASK13 or other tagged versions that can be detected using antibodies against the tag rather than directly against ASK13 .

For developing detection methods, researchers working with ASK13 typically use one of these approaches:

• Generation of peptide antibodies against unique regions of ASK13

• Expression of recombinant ASK13 in bacterial systems followed by purification and antibody production

• Creation of tagged versions (GFP, His-tag) of ASK13 that can be detected using commercial antibodies against the tag

How should ASK13 antibodies be validated for specificity?

Validation of ASK13 antibodies should follow a multi-step approach to ensure specificity:

• Western blot analysis: Compare protein detection in wild-type plants versus ASK13 overexpression lines and ASK13 knockdown/knockout lines. A specific antibody will show enhanced signals in overexpression lines and reduced signals in knockdown lines .

• Immunoprecipitation followed by mass spectrometry: Confirm that the immunoprecipitated protein is indeed ASK13.

• Peptide competition assay: Pre-incubation of the antibody with the immunizing peptide should abolish the signal if the antibody is specific.

• Cross-reactivity testing: Test the antibody against related ASK proteins to ensure it doesn't cross-react with other members of the SKP1 family.

• Immunohistochemistry controls: When performing tissue localization studies, include appropriate negative controls (secondary antibody only) and peptide competition controls .

What are the recommended protocols for isolating and cloning ASK13?

For efficient isolation and cloning of ASK13, the following detailed protocol is recommended:

• RNA isolation: Extract total RNA from Arabidopsis seeds using a modified method as described by Singh et al. (2003). This is particularly important as ASK13 is differentially expressed during seed development .

• cDNA synthesis: Prepare cDNA using a commercial cDNA synthesis kit (such as those from Applied Biosystems) with random primers and DNase I-treated RNA (approximately 2 μg) .

• PCR amplification: Amplify the full-length cDNA of ASK13 using gene-specific primers designed based on the gene sequence data available in The Arabidopsis Information Resource (TAIR) .

• Cloning: Clone the amplified product initially into a general cloning vector (such as pJET1.2) for sequence verification, followed by sub-cloning into appropriate expression vectors depending on experimental needs .

• Vector selection: For protein expression studies, consider using Gateway-compatible vectors that facilitate easy transfer between different expression systems .

How can researchers generate ASK13 overexpression and knockdown lines?

Generation of ASK13 modified expression lines involves these methodological steps:

For overexpression lines:

• Amplify full-length ASK13 cDNA using gene-specific primers and clone into an entry vector (e.g., pJET1.2) .

• Subclone ASK13 downstream of a constitutive promoter (such as CaMV35S) in a binary vector (e.g., pCAMBIA2301) .

• For fusion protein studies, clone ASK13 into intermediate vectors (such as pKYLX80:GFP) and then transfer the fusion construct to the binary vector .

• Transform the final construct into Agrobacterium tumefaciens followed by Arabidopsis transformation using the floral dip method .

• Select transformants on appropriate antibiotic-containing media and confirm overexpression through transcript analysis .

For knockdown lines:

• Identify unique sequences in ASK13 cDNA (including the 3'-UTR) as targets for RNA interference .

• Amplify these sequences using specific primers and clone into an entry vector .

• Transfer to a Gateway-compatible RNAi vector like pHELLSGATE12, which creates hairpin RNA constructs .

• Transform plants as described for overexpression lines and confirm knockdown efficiency through transcript analysis .

What methods are effective for studying ASK13 protein-protein interactions?

The search results highlight several complementary approaches for investigating ASK13 protein interactions:

• Yeast Two-Hybrid (Y2H) screening:

  • Use Gateway-compatible vectors (pDEST-GBKT7 and pDEST-GADT7) for constructing bait and prey plasmids .

  • Transform ASK13 bait construct into Y2H gold strain and mate with a normalized Arabidopsis Y2H cDNA library .

  • Select positive interactions on appropriate selective media lacking histidine, tryptophan, leucine, and adenine, supplemented with X-α-Gal and aureobasidin A .

  • Validate potential interactions through one-to-one Y2H assays .

• Bimolecular Fluorescence Complementation (BiFC):

  • Clone ASK13 into pSAT4-DEST-N (1–174) EYFP-C1 vector and potential interacting proteins into pSAT5-DEST-C (175-END) EYFP-C1 vector .

  • Co-transform the constructs into onion epidermal cells through particle bombardment .

  • Visualize interactions through fluorescence microscopy; a positive interaction reconstitutes the split YFP, producing fluorescence .

• Co-immunoprecipitation (Co-IP):

  • Express tagged versions of ASK13 and potential interacting partners.

  • Extract proteins under non-denaturing conditions to preserve interactions.

  • Use antibodies against the tag to pull down protein complexes.

  • Analyze co-precipitated proteins by immunoblotting or mass spectrometry.

How can researchers determine the oligomeric state of ASK13 protein?

The search results describe sophisticated approaches to determine ASK13's oligomeric state:

• Size-exclusion chromatography:

  • Express ASK13 as a C-terminal histidine-tagged fusion protein in E. coli BL-21(DE3) cells .

  • Induce expression with 0.5 mM IPTG for 8 hours at 25°C .

  • Purify the soluble protein using nickel-charged affinity columns .

  • Perform size-exclusion chromatography to determine the native molecular mass and potential oligomeric states .

  • For plant-expressed protein, extract GFP-fused ASK13 from transgenic Arabidopsis seedlings using appropriate extraction buffer (100 mM HEPES buffer, pH 7.5, 1 mM β-mercaptoethanol, and protease inhibitor cocktail) .

• Analytical ultracentrifugation:

  • This technique, though not explicitly mentioned in the search results, can provide definitive data on the oligomeric state and shape of proteins in solution.

  • Purified ASK13 can be analyzed at different concentrations to determine concentration-dependent oligomerization.

The search results indicate that ASK13 exists not only as a monomer but also as a homo-oligomer or heteromer with other ASK proteins, suggesting complex regulatory mechanisms .

What approaches can be used to study ASK13 expression patterns in different plant tissues?

For comprehensive analysis of ASK13 expression patterns, researchers should employ multiple complementary techniques:

• Promoter-GUS fusion analysis:

  • Clone the ASK13 promoter region upstream of the β-glucuronidase (GUS) reporter gene .

  • Transform Arabidopsis plants with this construct .

  • Perform GUS staining on various tissues (seedlings, leaves, flowers, siliques, and seeds) by incubating overnight in GUS staining solution (100 mM phosphate buffer (pH 7.0), 1 mM X-gluc, 10 mM EDTA, 0.1% (v/v) Triton X-100) at 37°C .

  • Remove chlorophyll with 95% ethanol and observe under a microscope .

• Real-time quantitative PCR (RT-qPCR):

  • Extract total RNA from different tissues and synthesize cDNA .

  • Perform RT-qPCR using ASK13-specific primers and appropriate endogenous controls (like 18S rRNA) .

  • Run reactions in triplicate with at least three biological replicates for statistical validity .

  • Normalize ASK13 expression to endogenous control genes for accurate comparison across tissues .

• Protein localization using GFP fusion:

  • Generate transgenic plants expressing ASK13-GFP fusion under native or constitutive promoters .

  • Observe GFP fluorescence in different tissues using confocal microscopy .

  • For subcellular localization, examine roots of transgenic seedlings using a laser confocal scanning microscope .

How does ASK13 function in response to abiotic stress conditions?

The search results indicate that ASK13 positively influences seed germination and seedling growth, particularly under abiotic stress conditions . To investigate this function in depth, researchers can implement these methodological approaches:

• Stress response phenotyping:

  • Compare wild-type, ASK13 overexpression, and ASK13 knockdown lines under various abiotic stress conditions (drought, salt, cold, heat).

  • Measure parameters such as germination rate, root length, fresh weight, and survival rate.

  • Document phenotypic differences through standardized photography protocols.

• Transcriptome analysis:

  • Perform RNA-seq on wild-type versus ASK13 modified lines under normal and stress conditions.

  • Identify differentially expressed genes and pathways affected by ASK13 expression levels.

  • Validate key findings using RT-qPCR on independent biological replicates.

• Protein interaction dynamics under stress:

  • Investigate if ASK13 interactions with partner proteins change under stress conditions.

  • Use co-immunoprecipitation or BiFC approaches under both normal and stress conditions.

  • Quantify changes in interaction strength or prevalence.

• Biochemical analysis of ASK13 modifications:

  • Examine if ASK13 undergoes post-translational modifications in response to stress.

  • Analyze phosphorylation, ubiquitination, or other modifications using mass spectrometry.

  • Determine if these modifications affect ASK13 function or interactions.

How can researchers address low signal problems when detecting ASK13 protein?

When facing challenges with ASK13 protein detection, consider these methodological solutions:

• Optimal protein extraction:

  • Use specialized extraction buffers for plant proteins (100 mM HEPES buffer, pH 7.5, 1 mM β-mercaptoethanol, and protease inhibitor cocktail) .

  • Grind tissue thoroughly in liquid nitrogen to ensure complete cell disruption.

  • Centrifuge at high speed (13,000 g for 15 min at 4°C) to remove cell debris .

  • Consider different extraction methods if ASK13 is associated with membranes or specific cellular compartments.

• Concentration of protein samples:

  • Use protein concentration methods (TCA precipitation, acetone precipitation) to increase protein concentration.

  • Load higher amounts of total protein when performing western blots.

• Enhanced detection methods:

  • Use high-sensitivity chemiluminescent substrates for western blotting.

  • Consider using biotin-streptavidin amplification systems.

  • Optimize antibody concentrations through titration experiments.

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

• Alternative detection approaches:

  • If direct detection is challenging, consider using epitope tags (His, GFP, FLAG) for indirect detection .

  • Express ASK13 as a fusion protein with a readily detectable tag .

What controls are essential when investigating ASK13 and its interactions?

Robust experimental design for ASK13 research requires these essential controls:

• For protein interaction studies:

  • Positive controls: Include known protein interaction pairs in Y2H or BiFC experiments .

  • Negative controls: Use pGADT7-T and pGBKT7-Lam transformed Y2H gold strain as negative controls for Y2H interactions .

  • Autoactivation controls: Test ASK13 bait construct alone to check for autoactivation in Y2H systems.

  • Empty vector controls: Include empty vectors in co-transformation experiments.

• For expression analysis:

  • No-RT controls: Include samples without reverse transcriptase to check for genomic DNA contamination .

  • Reference gene controls: Use stable reference genes (like 18S rRNA) for normalization in qPCR experiments .

  • Biological replicates: Perform experiments with at least three biological replicates for statistical validity .

• For functional studies:

  • Wild-type controls: Always compare transgenic lines with wild-type plants grown under identical conditions.

  • Vector-only controls: Include plants transformed with empty vectors.

  • Complementation controls: Perform genetic complementation by introducing functional ASK13 into knockdown/knockout lines .

How can researchers differentiate between direct and indirect effects of ASK13 on observed phenotypes?

Distinguishing direct from indirect effects requires sophisticated experimental approaches:

• Temporal analysis of gene expression:

  • Perform time-course experiments after inducing ASK13 expression or stress conditions.

  • Identify primary (early) versus secondary (late) responsive genes.

  • Use inducible promoter systems to control ASK13 expression timing precisely.

• Direct target identification:

  • Perform chromatin immunoprecipitation (ChIP) if ASK13 might directly affect transcription.

  • Use protein interaction data to identify direct binding partners versus downstream effectors.

  • Employ rapid biochemical assays that can detect immediate protein modifications or interactions.

• Genetic approaches:

  • Create double mutants combining ASK13 mutants with mutants of potential downstream factors.

  • Epistasis analysis can help establish the order of gene action in a pathway.

  • Suppress individual suspected downstream pathways and observe if ASK13 effects persist.

• Domain-specific mutations:

  • Generate ASK13 variants with mutations in specific functional domains.

  • Determine which domains are essential for particular phenotypes or interactions.

  • This approach can separate different functions of the ASK13 protein.