pmk-3 Antibody
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
How do I validate antibody specificity for PMK-3 in C. elegans studies?
Methodological approach:
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Use pmk-3 knockout mutants as negative controls in immunofluorescence assays .
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Combine RNA interference (RNAi) targeting pmk-3 with Western blotting to confirm signal reduction .
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Cross-validate with phosphorylation-specific antibodies (e.g., targeting Thr/Tyr residues in the activation loop) to distinguish active PMK-3 from inactive forms .
Key challenge: PMK-3 shares structural homology with PMK-1/PMK-2, necessitating rigorous specificity testing .
What experimental models best demonstrate PMK-3's role in axonal regeneration?
Advanced research design:
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Use syp-2(ok307) mutants with persistent double-strand breaks (DSBs) to study PMK-3's interaction with synaptonemal complex proteins .
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Employ tissue-specific RNAi in the intestine or neurons to dissect somatic vs. germline signaling roles .
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Monitor phosphorylation cascades using recombinant PMK-3 and MAK2 kinase assays to map activation dynamics .
Table 1: PMK-3 functional assays
| Assay Type | Readout | Key Mutants Used |
|---|---|---|
| Kinase Activity | MAK2 phosphorylation | dlk-1 (MAP3K knockout) |
| Apoptosis Quantification | Germ cell counts | syp-2; pmk-3 double mutants |
| Stress Response | qRT-PCR for egl-1, ced-13 | pmk-1(km25) intestinal rescue |
How to resolve contradictory data on PMK-3's somatic vs. germline roles?
Data contradiction analysis:
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Scenario: Discrepancies in apoptosis regulation (germline vs. somatic tissues) .
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Solution:
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Perform tissue-specific promoter-driven PMK-3 overexpression (e.g., ges-1 for intestine, rab-3 for neurons) .
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Compare phosphorylation patterns of PMK-3 in cep-1/p53 mutants to isolate DNA damage response pathways .
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Use dual immunofluorescence for p-PMK-3 and germline markers (e.g., HIM-4) to localize activity .
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What are optimal conditions for detecting PMK-3 activation under oxidative stress?
Experimental optimization:
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Treat worms with 50 Gy ionizing radiation (IR) and harvest samples at 2-hour post-exposure for peak phosphorylation .
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Use lysis buffers with phosphatase inhibitors (e.g., sodium orthovanadate) to preserve phosphorylation states .
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Validate stress induction via parallel assays for SOD-3::GFP reporters or mitochondrial ROS levels .
How does PMK-3 differ functionally from PMK-1/PMK-2 isoforms?
Comparative analysis:
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Expression: PMK-3 is enriched in neurons, while PMK-1 localizes to the intestine .
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Pathways: PMK-3 regulates DLK-1/MKK-4-dependent axonal regeneration, unlike PMK-1’s role in pathogen response .
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Structural distinction: PMK-3 lacks the C-terminal docking motif found in PMK-1, altering substrate specificity .
What controls are critical for PMK-3 antibody-based assays?
Quality assurance steps:
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Include pmk-3 null mutants (e.g., CRISPR-generated deletions) in every blot/IF experiment .
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Pre-absorb antibodies with recombinant PMK-3 protein to test nonspecific binding .
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Validate cross-reactivity using heterologous systems (e.g., HEK293T cells expressing PMK-1/PMK-2/PMK-3) .
How to study PMK-3's interaction with downstream effectors like MAK2?
Advanced methodology:
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Co-immunoprecipitation (Co-IP) with anti-PMK-3 and anti-MAK2 antibodies in lysates from IR-treated worms .
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In vitro kinase assays using purified PMK-3 and MAK2, with ATP-γ-S for thiophosphorylation labeling .
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FRET-based biosensors to visualize real-time PMK-3-MAK2 interaction dynamics in neuronal cells .
