PIE1 Antibody
Description
Definition and Target Specificity
The PIE1 Antibody is a polyclonal or monoclonal immunoglobulin raised against epitopes of the PIE-1 protein, a 68-kDa CCCH zinc finger protein. It binds to PIE-1 with high specificity, enabling detection via:
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Immunofluorescence: Localization in germline nuclei and P granules .
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Western blot: Verification of protein expression levels during embryogenesis .
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Immunoprecipitation: Identifying interactors like SUMO components (e.g., UBC-9, GEI-17) and histone deacetylases (HDA-1) .
Applications in Research
The PIE1 Antibody is pivotal for dissecting PIE-1’s multifaceted roles:
Dual Functional Domains
PIE-1’s bifunctional activity is mediated by distinct structural regions:
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Transcriptional Repression:
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Maternal RNA Activation:
Immunostaining Workflow
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Fixation: 4% formaldehyde in PBS.
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Permeabilization: 0.1% Triton X-100.
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Primary Antibody: Anti-PIE-1 (1:200 dilution, overnight at 4°C).
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Secondary Antibody: Alexa Fluor-conjugated (1:500, 1 hr at RT).
Western Blot Optimization
SUMO-Dependent Germline Maintenance
Studies using PIE1 Antibody revealed:
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Embryonic Germline: PIE-1 interacts with SUMO E3 ligases (GEI-17) to suppress somatic differentiation .
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Adult Germline: SUMOylation of PIE-1 at K68 activates HDA-1, reducing histone H3K9 acetylation and silencing transposons (e.g., Tc5, MIRAGE1) .
Chromatin Remodeling
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HDA-1 Recruitment: PIE-1 binds HDA-1 and LET-418 (Mi-2 homolog), promoting nucleosome remodeling .
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Gene Expression Profiling: pie-1::degron depletion upregulates 479 genes, including spermatogenesis genes and transposons .
Future Directions
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Therapeutic Targets: Exploring PIE-1’s role in cancer stem cells, given its parallels to human germ cell malignancies.
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Cross-Species Validity: Investigating PIE-1 homologs in mammals for conserved SUMO-HDAC pathways.
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Single-Cell Analysis: High-resolution mapping of PIE-1 gradients during embryogenesis.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Studies indicate that mutations in genes encoding SWR1 complex subunits, namely photoperiod-independent Early Flowering1 (PIE1), actin-related protein6 (ARP6), and SWR1 complex6 (SWC6), lead to increased sensitivity to various DNA damaging agents. PMID: 23780875
- The discovery and characterization of SERRATED LEAVES AND EARLY FLOWERING (SEF) in A. thaliana and its interactions with other proteins, including ATARP6 and PIE1, have been reported. PMID: 17142478
- Arabidopsis SWC6 (AtSWC6), SUPPRESSOR OF FRIGIDA 3 (SUF3), and PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 (PIE1) are homologs of SWC6, ARP6, and SWR1, respectively. These proteins have roles in development, including floral repression through the full activation of FLOWERING LOCUS C. PMID: 17470967
Q&A
Basic Research Questions
How do I validate PIE-1 antibody specificity for immunocytochemistry in C. elegans?
Validation requires a multi-step approach:
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Knockout/RNAi controls: Compare staining in wild-type vs. PIE-1-depleted embryos. Absence of signal in mutants confirms specificity .
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Pre-adsorption control: Pre-incubate the antibody with recombinant PIE-1 protein to block epitope binding .
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Subcellular localization: Verify nuclear and P granule localization patterns using confocal microscopy, as cytoplasmic P granule association is a hallmark of PIE-1 .
Table 1: Key validation steps for PIE-1 antibodies
| Control Type | Method | Expected Outcome |
|---|---|---|
| Knockout | Compare wild-type vs. mutant embryos | No signal in mutants |
| Pre-adsorption | Antibody + excess antigen | Reduced/abolished staining |
| Secondary antibody control | Omit primary antibody | No signal |
What are the primary applications of PIE-1 antibodies in developmental biology?
PIE-1 antibodies are critical for:
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Germline fate studies: Tracking PIE-1’s nuclear exclusion in somatic blastomeres via immunofluorescence .
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Transcriptional repression assays: Co-staining with RNA polymerase II phosphorylation markers (e.g., Ser5-CTD) to confirm inhibition .
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SUMOylation analysis: Detecting SUMO-conjugated PIE-1 using immunoprecipitation followed by Western blot .
Advanced Research Questions
How can conflicting data on PIE-1’s role in transcriptional repression be resolved?
Discrepancies often arise from:
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Experimental models: Embryonic vs. adult germline contexts (e.g., PIE-1 inhibits transcription in embryos but promotes chromatin remodeling in adults) .
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Antibody cross-reactivity: Validate using pie-1 null mutants and test for off-target binding to paralogs like MEP-1 .
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Technical variability: Optimize fixation protocols (e.g., methanol vs. formaldehyde) to preserve nuclear PIE-1 foci .
Table 2: Common contradictions and solutions
| Issue | Resolution | Source Support |
|---|---|---|
| Variable nuclear localization | Use high-resolution imaging (SIM/STED) | |
| Inconsistent SUMOylation | Combine IP with SUMO protease treatment |
What advanced methods elucidate PIE-1’s interaction with SUMOylation machinery?
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Yeast two-hybrid screens: Identify physical interactions (e.g., PIE-1 binds SMO-1/SUMO and UBC-9) .
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In vitro SUMOylation assays: Incubate recombinant PIE-1 with SUMO E1/E2 enzymes and ATP, then detect shifts via Western blot .
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Genetic epistasis: Test synthetic lethality in pie-1; smo-1 double mutants .
How to optimize PIE-1 antibody concentration for chromatin immunoprecipitation (ChIP)?
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Titration series: Test 0.1–2 µg/mL antibody in crosslinked embryos, using pie-1 mutants as negative controls.
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Crosslinking efficiency: Compare formaldehyde vs. DSG crosslinkers to preserve DNA-protein interactions .
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Spike-in controls: Add Drosophila chromatin with exogenous PIE-1 as an internal standard .
Methodological Best Practices
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Batch consistency: Use monoclonal antibodies (e.g., Proteintech 15,939‐1‐AP) to minimize variability.
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Multiplex imaging: Combine PIE-1 staining with P granule markers (e.g., PGL-1) for co-localization studies .
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Quantitative analysis: Use ImageJ/Fiji plugins to quantify nuclear vs. cytoplasmic PIE-1 fluorescence intensity .
