GASA14 Antibody

What is GASA14 and what biological functions does it serve?

GASA14 is a member of the GASA (GA-stimulated in Arabidopsis) family of proteins that plays significant roles in plant development and stress responses. GASA14 expression is upregulated by gibberellic acid (GA) and downregulated by DELLA proteins GAI and RGA, which are transcriptional regulators that repress GA responses . The protein contains both GASA and proline-rich protein (PRP) domains, with the GASA domain being essential for its biological functions .

Functionally, GASA14 regulates leaf expansion in young plants, with phenotypic analysis showing retarded growth in GASA14 null mutant (gasa14-1) lines and promoted growth in 35S::GASA14 overexpression lines . Additionally, GASA14 is involved in GA-dependent responses, as demonstrated by the increased sensitivity of gasa14-1 plants to paclobutrazol (an inhibitor of GA biosynthesis) . Perhaps most significantly, GASA14 contributes to abiotic stress resistance by modulating reactive oxygen species (ROS) accumulation, providing enhanced tolerance to abscisic acid (ABA) and salt stress .

How do I select the appropriate GASA14 antibody for my research?

When selecting a GASA14 antibody for your research, consider the following methodological approach:

• Define your experimental goals: Determine which techniques you'll be using (Western blot, immunoprecipitation, immunofluorescence, etc.) as different antibodies may perform optimally in different applications.

• Consider antibody type: While polyclonal antibodies offer high sensitivity and recognize multiple epitopes (beneficial for detecting proteins in native states), monoclonal antibodies provide greater specificity and consistency between batches.

• Verify species reactivity: Ensure the antibody recognizes GASA14 in your species of interest. While GASA14 is well-characterized in Arabidopsis, cross-reactivity with other plant species should be validated.

• Review validation data: Examine the manufacturer's validation data, including Western blot results showing the expected molecular weight band for GASA14, absence of non-specific binding, and performance in relevant applications.

• Consider positive controls: Generate or obtain GASA14 overexpression lines (such as 35S::GASA14) to serve as positive controls for antibody validation .

What are the most effective techniques for GASA14 protein detection?

Several techniques are effective for GASA14 protein detection in plant tissues:

• Western blot analysis: This remains the gold standard for protein detection. Follow protocols similar to those used for related proteins, where 10μg of protein per sample is loaded, and the membrane is incubated with purified antibody followed by detection using ECL reagents .

• Immunoprecipitation (IP): IP can be used to isolate GASA14 from complex protein mixtures. Typically, purified antibodies are added to protein extracts along with protein A/G beads, followed by overnight incubation at 4°C .

• Co-immunoprecipitation (CoIP): To study protein-protein interactions involving GASA14, CoIP can be performed using methodologies similar to those applied for other plant proteins, involving protein extraction, antibody incubation, and precipitation with protein A beads .

• Immunofluorescence: For tissue localization, paraffin sections can be dewaxed, subjected to antigen retrieval, incubated with GASA14 antibody overnight at 4°C, followed by secondary antibody incubation and visualization under fluorescence microscopy .

• ELISA: For quantitative assessment of GASA14 levels, various ELISA formats can be employed depending on the research question.

What controls should I include when working with GASA14 antibody?

When working with GASA14 antibody, include these essential controls:

• Positive controls:

  • Plant tissues known to express GASA14 (e.g., young Arabidopsis leaves)

  • Transgenic 35S::GASA14 lines with overexpressed GASA14

  • Recombinant GASA14 protein (if available)

• Negative controls:

  • GASA14 null mutant (gasa14-1) tissues

  • Pre-immune serum (for polyclonal antibodies)

  • Primary antibody omission

  • Isotype controls (for monoclonal antibodies)

• Specificity controls:

  • Peptide competition assay, where the antibody is pre-incubated with the immunizing peptide

  • Testing on related GASA family proteins to confirm specificity

• Loading controls:

  • Housekeeping proteins like Elongation factor 1 alpha-like (EF1αL) for normalization in Western blots

  • Coomassie staining of replicate gels

Implementing these controls ensures experimental validity and helps troubleshoot issues with antibody specificity or sensitivity.

How can I investigate GASA14's role in ROS modulation using antibody-based techniques?

To investigate GASA14's role in ROS modulation, implement this methodological approach:

• Comparative protein localization during oxidative stress:

  • Perform immunofluorescence microscopy on wild-type, GASA14 null mutant (gasa14-1), and overexpression (35S::GASA14) plant tissues subjected to oxidative stress conditions .

  • Co-stain with ROS indicators (such as DCF-DA or NBT) to correlate GASA14 localization with ROS accumulation sites.

  • Analyze subcellular redistribution of GASA14 upon H₂O₂ or other ROS inducers treatment.

• Protein-protein interaction analysis:

  • Use co-immunoprecipitation with GASA14 antibody to pull down interaction partners under normal and oxidative stress conditions .

  • Combine with mass spectrometry to identify novel binding partners that may be involved in ROS signaling pathways.

  • Validate interactions using techniques such as yeast two-hybrid or split luciferase complementation assays .

• Post-translational modification assessment:

  • Investigate whether oxidative stress induces post-translational modifications of GASA14 through immunoprecipitation followed by mass spectrometry.

  • Use phospho-specific antibodies (if available) to determine if GASA14 phosphorylation status changes during oxidative stress.

• Chromatin immunoprecipitation (ChIP) analysis:

  • If GASA14 has nuclear localization, perform ChIP to identify potential DNA binding sites and target genes involved in ROS metabolism.

• Quantitative changes in GASA14 expression:

  • Use Western blot combined with densitometry to quantify changes in GASA14 protein levels during various oxidative stress conditions .

  • Compare results with gene expression data to identify post-transcriptional regulation mechanisms.

These approaches will provide comprehensive insights into how GASA14 modulates ROS accumulation and contributes to stress resistance in plants.

What are the optimal parameters for immunohistochemical detection of GASA14 in different plant tissues?

For optimal immunohistochemical detection of GASA14 in different plant tissues, follow these methodological guidelines:

• Tissue fixation and processing:

  • Use a fixing solution containing 2% paraformaldehyde and 2% polyvinylpyrrolidone 40 with 10 mM dithiothreitol (pH 7.0) at 4°C for 1 hour .

  • For woody tissues, extend fixation time and consider vacuum infiltration to ensure complete penetration.

  • Process tissues through dehydration and paraffin embedding with gradual transitions to prevent tissue damage.

• Antigen retrieval optimization:

  • Test different antigen retrieval methods, including heat-induced epitope retrieval using EDTA buffer (pH 8.0) .

  • For recalcitrant tissues, enzymatic antigen retrieval with proteinase K may be necessary.

  • Optimize microwave heating times and power settings based on tissue type.

• Antibody concentration and incubation conditions:

  • Perform titration experiments with different antibody dilutions (1:100 to 1:2000).

  • Test both overnight incubation at 4°C and shorter incubations (2-4 hours) at room temperature.

  • Evaluate different blocking agents (3% BSA, normal serum, or commercial blocking solutions) to minimize background .

• Signal detection optimization:

  • For fluorescence detection, evaluate different secondary antibodies and fluorophores.

  • Use spontaneous fluorescence quenching reagent to reduce autofluorescence, particularly in tissues with high chlorophyll content .

  • For chromogenic detection, compare different peroxidase substrates (DAB, AEC) for optimal signal-to-noise ratio.

• Tissue-specific considerations:

  • For root tissues: Extend permeabilization time and consider using higher antibody concentrations.

  • For leaf tissues: Implement additional steps to reduce chlorophyll autofluorescence.

  • For meristematic regions: Use thinner sections (5-7 μm) for better antibody penetration.

• Controls and validation:

  • Include wild-type, GASA14 null mutant (gasa14-1), and overexpression (35S::GASA14) tissues as controls .

  • Perform peptide competition assays to confirm antibody specificity in different tissues.

These optimized parameters will ensure reliable and reproducible immunohistochemical detection of GASA14 across various plant tissues.

How can I use GASA14 antibody to investigate protein-protein interactions in stress response pathways?

To investigate GASA14 protein-protein interactions in stress response pathways, implement these methodological approaches:

• Co-immunoprecipitation (CoIP) with stress-specific modifications:

  • Extract total protein from plants subjected to different stress treatments (ABA, salt, oxidative stress) .

  • Use purified anti-GASA14 antibody for immunoprecipitation, followed by Western blot analysis with antibodies against potential interacting partners .

  • Compare interaction profiles between normal and stress conditions to identify stress-specific interactions.

  • Protocol parameters: Use 10 μl of purified antibody with 20 μl of protein A beads, incubate overnight at 4°C, wash thoroughly, and analyze by SDS-PAGE .

• Proximity-dependent biotin identification (BioID):

  • Generate transgenic plants expressing GASA14 fused to a promiscuous biotin ligase.

  • Expose plants to stress conditions, then purify biotinylated proteins using streptavidin beads.

  • Identify proximal proteins by mass spectrometry and validate using CoIP with GASA14 antibody.

• Split luciferase complementation assay validation:

  • After identifying potential interactors, validate using split luciferase complementation assays.

  • Clone GASA14 and candidate interactors into pCAMBIA1-cLUC and pCAMBIA1-nLUC vectors .

  • Compare interaction strength under normal and stress conditions by measuring luminescence intensity.

• Yeast two-hybrid screening with stress-related conditions:

  • Use GASA14 as bait in yeast two-hybrid screens with cDNA libraries from stressed plant tissues.

  • Clone full-length GASA14 into pGBKT7 vector and screen against stress-induced cDNA libraries in pGADT7 .

  • Validate positive interactions by CoIP with GASA14 antibody in plant tissues.

• Antibody-based protein array analysis:

  • Develop protein arrays containing stress-related proteins.

  • Probe with biotinylated GASA14 protein, followed by detection systems.

  • Compare binding profiles under different stress conditions.

• Domain-specific interaction mapping:

  • Generate antibodies against specific domains of GASA14 (GASA domain versus PRP domain).

  • Perform domain-specific CoIP to determine which domain mediates specific protein interactions.

  • This approach can reveal whether the GASA domain, which is necessary for GASA14 functions , is also the primary mediator of protein interactions.

These methodologies will provide comprehensive insights into how GASA14 interactions change during stress responses and identify key partners in stress signaling pathways.

How does GASA14 compare with other GASA family proteins in terms of antifungal activity, and how can antibody-based techniques help characterize this?

While specific data on GASA14's antifungal activity is limited in the provided search results, we can draw insights from related GASA proteins and outline antibody-based approaches to characterize this activity:

• Comparative activity assessment:

  • Based on data from TdGASA1 (a durum wheat GASA protein), GASA family proteins can exhibit significant antifungal activity against various fungal pathogens including Aspergillus and Fusarium species .

  • TdGASA1 showed inhibition zones of 17-22 mm against fungal strains with MIC values ranging from 0.312 to 1.25 mg/mL .

• Antibody-based methodologies to characterize GASA14 antifungal activity:

  a. Immunodepletion assays:

  • Use GASA14 antibodies to deplete the protein from plant extracts.

  • Compare antifungal activity of normal and immunodepleted extracts to determine GASA14's contribution.

  • Reconstitute activity by adding purified GASA14 protein.

  b. Localization during pathogen infection:

  • Perform immunofluorescence studies using GASA14 antibodies on plant tissues during fungal infection.

  • Determine if GASA14 localizes to infection sites or accumulates in tissues responding to pathogen attack.

  • Compare localization patterns between wild-type and 35S::GASA14 plants with enhanced resistance .

  c. Protein expression kinetics:

  • Use Western blot analysis with GASA14 antibodies to monitor protein accumulation during pathogen infection.

  • Correlate GASA14 expression levels with resistance phenotypes.

  • Compare expression kinetics across different GASA family members using specific antibodies.

  d. Structural integrity assessment:

  • Use antibodies to confirm structural integrity of GASA14 after extraction and purification.

  • Ensure that antifungal activity assays are conducted with properly folded protein.

  • Perform epitope mapping to determine which regions are critical for activity.

• Mechanism investigation:

  • GASA proteins may exert antioxidant properties through their cysteine-rich domains, participating in H₂O₂ elimination .

  • Overexpression of GASA14 in Arabidopsis leads to decreased ROS production , which may contribute to antifungal resistance.

  • Use GASA14 antibodies in combination with ROS detection to investigate whether GASA14's antifungal activity is related to its ROS-modulating function .

These approaches would help characterize GASA14's potential antifungal activity and compare it with other GASA family proteins like TdGASA1.

What techniques can reveal the molecular mechanism of GASA14's role in abiotic stress resistance?

To elucidate the molecular mechanism of GASA14's role in abiotic stress resistance, implement these antibody-dependent methodological approaches:

• Temporal and spatial protein dynamics analysis:

  • Use Western blot analysis with GASA14 antibody to track protein levels during stress progression .

  • Perform immunofluorescence microscopy to determine subcellular localization changes upon stress exposure .

  • Compare GASA14 protein accumulation patterns between normal conditions and various abiotic stresses (ABA treatment, salt stress, drought) .

• Post-translational modification profiling:

  • Immunoprecipitate GASA14 using specific antibodies from stressed and unstressed plants.

  • Analyze by mass spectrometry to identify stress-induced post-translational modifications.

  • Develop or obtain modification-specific antibodies (phospho, redox-sensitive) to track specific GASA14 modifications during stress responses.

• Redox state analysis:

  • Since GASA14 modulates ROS accumulation , perform redox proteomics after immunoprecipitation.

  • Use antibodies against oxidized cysteines to determine if GASA14's cysteine-rich domain undergoes redox changes during stress.

  • Compare redox states between wild-type and 35S::GASA14 lines that show enhanced stress resistance .

• Protein complex dynamics:

  • Perform Blue Native-PAGE followed by Western blot using GASA14 antibodies to identify stress-responsive protein complexes.

  • Use size exclusion chromatography coupled with immunodetection to monitor changes in GASA14-containing complexes during stress.

  • Implement CoIP under normal and stress conditions to identify stress-specific interaction partners .

• Domain-specific function analysis:

  • Generate domain-specific antibodies against the GASA and PRP domains.

  • Since the GASA domain is necessary for GASA14 functions , use domain-specific antibodies to determine which domain interacts with specific partners during stress.

  • Combine with domain-deletion mutants and functional complementation assays.

• Chromatin association and transcriptional regulation:

  • If GASA14 has nuclear localization, perform ChIP using GASA14 antibodies to identify potential DNA binding sites.

  • Compare chromatin binding profiles under normal and stress conditions.

  • Correlate with transcriptomic data to identify GASA14-regulated stress-responsive genes.

• ROS-specific mechanism analysis:

  • Combine GASA14 immunodetection with in situ ROS visualization techniques.

  • Implement protein-protein interaction assays to identify if GASA14 directly interacts with ROS-producing or ROS-scavenging enzymes.

  • Measure enzymatic activities of antioxidant enzymes (CAT, POD, SOD) in immunoprecipitates from GASA14 complexes .

These approaches will provide comprehensive insights into how GASA14 mediates abiotic stress resistance through ROS modulation and other potential mechanisms.