At4g40100 Antibody
Description
Definition and Target Protein
The At4g40100 antibody recognizes the PRSL1 protein, a regulatory subunit of Protein Phosphatase 1 (PP1c) in Arabidopsis . PP1c is a serine/threonine phosphatase involved in dephosphorylating key proteins, including the plasma membrane H+-ATPase essential for stomatal opening . PRSL1 binds PP1c via its RVxF motif, facilitating blue light-induced phosphorylation of H+-ATPase in guard cells .
Functional Role in Stomatal Regulation
Research demonstrates PRSL1’s involvement in phototropin-mediated stomatal opening:
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Genetic Evidence: prsl1 mutants exhibit impaired blue light-dependent stomatal opening, reduced H+ pumping, and decreased phosphorylation of H+-ATPase compared to wild-type plants .
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Mechanistic Insight: PRSL1 integrates signals from phototropins (phot1/phot2) to activate PP1c, which dephosphorylates the autoinhibitory domain of H+-ATPase, enabling proton extrusion and stomatal aperture .
Photoperiodic Regulation and Gene Expression
At4g40100 expression is modulated by photoperiod conditions:
| AGI No. | Name | SS (Short-Day) | SL (Long-Day) | Fold Change (SL/SS) | P-value |
|---|---|---|---|---|---|
| AT4g40100 | Hypothetical protein | 1871 ± 140 | 4155 ± 627 | 2.28 ± 0.43 | 0.024 |
This 2.28-fold upregulation under long-day conditions suggests a role in photoperiodic flowering or circadian responses .
Research Applications
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Stomatal Physiology: Used to investigate PP1c-PRSL1 interactions in guard cell signaling .
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Protein Localization: Identifies PRSL1 expression patterns in response to light and hormonal stimuli.
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Mutant Validation: Confirms PRSL1 knockout phenotypes in stomatal assays .
Key Findings from Peer-Reviewed Studies
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PP1c Binding: PRSL1’s RVxF motif is critical for PP1c interaction, as shown via yeast two-hybrid assays .
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Cross-Talk with Phototropins: PRSL1 mutants retain functional phototropin kinase activity but fail to activate H+-ATPase, indicating downstream signaling defects .
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Evolutionary Conservation: Homologs of PRSL1 exist in Vicia faba, suggesting conserved PP1 regulatory mechanisms in plants .
Implications for Plant Biology
The At4g40100 antibody provides tools to dissect:
Product Specs
Q&A
Basic Research Questions
What experimental strategies are recommended for validating At4g40100 antibody specificity in Arabidopsis thaliana studies?
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Methodological Answer:
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Knockout/Wild-Type Comparisons: Use Arabidopsis knockout mutants for At4g40100 (if available) alongside wild-type plants. Perform Western blot (WB) to confirm absence of signal in mutants .
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Peptide Competition Assays: Pre-incubate the antibody with recombinant At4g40100 protein. Loss of signal in WB/ELISA confirms specificity .
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Cross-Species Testing: Validate reactivity against non-Arabidopsis plant extracts to rule out nonspecific binding .
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| Validation Method | Key Outcome | Limitations |
|---|---|---|
| Knockout Line Analysis | Specificity confirmed by absent signal | Requires mutant availability |
| Recombinant Protein Blocking | Signal reduction indicates specificity | Dependent on protein purity |
How should At4g40100 antibodies be stored and handled to ensure stability in long-term experiments?
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Methodological Answer:
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Storage: Aliquot and store at -20°C (short-term) or -80°C (long-term) in 50% glycerol/PBS buffer to prevent freeze-thaw degradation .
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Buffer Composition: Contains 0.03% Proclin 300 preservative, which minimizes bacterial growth .
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Reconstitution: Centrifuge vials briefly before use to recover settled antibodies.
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| Condition | Recommendation |
|---|---|
| Temperature | -20°C (stable for months) / -80°C (years) |
| Freeze-Thaw Cycles | ≤3 cycles to prevent aggregation |
What are the best practices for optimizing At4g40100 antibody dilution in ELISA and Western blot?
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Methodological Answer:
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Serial Dilution Testing: Start with 1:500–1:2000 for WB and 1:1000–1:5000 for ELISA, adjusting based on signal-to-noise ratio .
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Negative Controls: Include secondary antibody-only lanes and nontransgenic plant extracts to identify background.
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Signal Enhancement: Use HRP-conjugated secondaries with chemiluminescent substrates for low-abundance targets.
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Advanced Research Questions
How can researchers address cross-reactivity concerns when using At4g40100 antibodies in mutant Arabidopsis lines?
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Methodological Answer:
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Epitope Mapping: Identify the antibody’s binding region using truncated recombinant proteins (e.g., N-terminal vs. C-terminal domains).
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CRISPR-Cas9 Rescue Lines: Reintroduce At4g40100 in knockout mutants to confirm antibody signal restoration.
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Mass Spectrometry Validation: Combine immunoprecipitation with LC-MS/MS to verify target identity in pulldown assays.
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What integrated approaches combine At4g40100 antibody-based protein detection with transcriptomic data?
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Methodological Answer:
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Correlative Analysis: Perform RNA-seq/qRT-PCR on tissues used for WB/immunohistochemistry (IHC) to compare mRNA and protein levels.
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Spatial Mapping: Use IHC-stained sections for laser-capture microdissection followed by RNA extraction.
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Co-Expression Networks: Overlay protein localization data (via antibody staining) with gene co-expression networks (e.g., ATTED-II database).
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| Integration Method | Application |
|---|---|
| RNA-Protein Correlation | Identifies post-transcriptional regulation |
| Spatial Transcriptomics | Links protein localization to gene expression |
How to troubleshoot discrepancies between At4g40100 antibody signals and phenotypic data in functional studies?
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Methodological Answer:
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Scenario 1: Strong WB signal but no observable phenotype.
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Action: Check for functional redundancy (e.g., paralogs) via phylogenetic analysis.
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Scenario 2: Phenotype observed but no antibody detection.
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Action: Verify antibody compatibility with post-translational modifications (e.g., phosphorylation, glycosylation).
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What emerging techniques enhance At4g40100 antibody utility in single-cell proteomics?
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Methodological Answer:
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CITE-Seq Adaptation: Conjugate antibodies to oligonucleotides for simultaneous protein and RNA detection in single cells.
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Nanoparticle-Assisted Imaging: Use gold-labeled antibodies for super-resolution microscopy in root tip cells.
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