pmo25 Antibody

What is PMO25 and why is an antibody against it valuable in research?

PMO25 is a phosphorodiamidate morpholino oligomer (PMO) antisense therapy designed to modulate splicing patterns of target genes, particularly in neuromuscular disorders like spinal muscular atrophy (SMA). It functions by binding to specific RNA sequences to alter pre-mRNA splicing, allowing exon skipping and restoration of reading frames in mutated genes . An antibody specifically recognizing PMO25 enables researchers to track morpholino distribution in tissues, validate delivery efficiency to target organs, and correlate treatment efficacy with morpholino presence in specific cell types through techniques like immunohistochemistry, Western blotting, and ELISA.

What experimental applications are most common for PMO25 antibody?

The PMO25 antibody is primarily utilized in the following research applications:

• Biodistribution studies: Detecting PMO25 localization across different tissues following administration, particularly in central nervous system (CNS) and muscle tissues

• Pharmacokinetic analyses: Measuring PMO25 persistence in tissues over time using Western blot quantification

• Correlative analyses: Associating PMO25 presence in tissues with molecular outcomes (e.g., exon skipping efficiency, SMN protein restoration)

• Optimization studies: Evaluating delivery methods by comparing PMO25 tissue penetration across different administration routes (intracerebroventricular vs. subcutaneous)

These applications are critical for understanding therapeutic mechanisms and optimizing treatment protocols in preclinical models of SMA and other neuromuscular disorders.

What detection methods are compatible with PMO25 antibody?

According to available product information, PMO25 antibody has been validated for the following detection methods:

• Western blotting (WB): For quantitative assessment of PMO25 in tissue lysates

• Enzyme-linked immunosorbent assay (ELISA): For high-throughput screening of PMO25 levels in biological samples

Additional applications common for similar research antibodies include:

• Immunohistochemistry (IHC): For visualizing tissue and cellular distribution of PMO25

• Immunofluorescence (IF): For co-localization studies with cell-type specific markers

Researchers should conduct preliminary validation experiments to optimize protocols for their specific experimental conditions.

How should samples be prepared for optimal PMO25 antibody detection?

While specific protocols may vary based on the particular antibody formulation, general recommendations for sample preparation include:

• Tissue fixation: Use of 4% paraformaldehyde for immunohistochemistry applications

• Tissue homogenization: For protein extraction, tissues should be homogenized in RIPA buffer supplemented with protease inhibitors

• Sample dilution: Serial dilutions are recommended when establishing optimal antibody concentrations

• Blocking conditions: BSA (3-5%) in PBS-T is typically effective for reducing background

• Incubation parameters: Primary antibody incubation at 4°C overnight typically yields optimal results

Researchers should consult specific antibody datasheets for optimized protocols and validate detection methods in their experimental systems.

How can PMO25 antibody help evaluate different delivery routes in therapeutic studies?

PMO25 antibody serves as a critical tool for comparing biodistribution and efficacy between different administration routes. Research has demonstrated significant differences in PMO25 efficacy between intracerebroventricular (ICV) and subcutaneous (SC) delivery methods. In SMA mouse models, SC delivery of high-dose (40 μg/g) PMO25 resulted in median survival of 261 days compared to 212 days with ICV delivery . This difference can be analyzed using PMO25 antibody to:

• Quantify tissue-specific distribution: Western blot analysis using PMO25 antibody can determine relative concentrations in CNS versus peripheral tissues

• Assess blood-brain barrier penetration: Immunohistochemistry can evaluate CNS penetration efficiency

• Analyze cellular uptake patterns: Microscopy with PMO25 antibody can identify specific cell populations efficiently taking up the morpholino

The table below summarizes comparative survival data between different delivery routes:

Data derived from Zhou et al., 2015

How does timing of administration affect PMO25 efficacy and what role does antibody detection play in optimization?

The therapeutic window for PMO25 administration has significant impact on treatment outcomes, which can be quantified using PMO25 antibody detection. Research demonstrates that:

• Early intervention is critical: Administration at postnatal day 0 (PND0) provides significantly better outcomes than delayed treatment

• Sequential dosing improves outcomes: Secondary low-dose (10 μg/g) administration at PND5 extended median survival from 22 to 92.5 days when delivered subcutaneously

• Therapeutic window narrows with disease progression: The efficacy of secondary administration decreased progressively when delayed to PND7 (78 days survival), PND10 (27 days) or PND12 (24 days)

PMO25 antibody enables researchers to:

• Track morpholino clearance rates to establish optimal redosing intervals

• Correlate tissue levels with functional outcomes at different time points

• Identify tissues with rapid versus sustained PMO25 retention

What methodological considerations are important when using PMO25 antibody for quantifying dose-response relationships?

When using PMO25 antibody to establish dose-response relationships, researchers should consider:

• Linear detection range: Validate the antibody's linear detection range using serial dilutions of purified PMO25 standards

• Sample normalization: Normalize PMO25 levels to total protein content or housekeeping proteins

• Cross-reactivity assessment: Test for potential cross-reactivity with endogenous oligonucleotides or other therapeutic oligomers

• Signal amplification: Consider signal amplification methods for detecting low PMO25 concentrations in tissues with limited uptake

Research has established clear dose-dependent effects of PMO25 treatment on SMN2 exon 7 inclusion and protein levels:

• 10 μg/g (low-dose) PMO25 treatment increased exon 7 inclusion 1.6-fold compared to untreated mice

• 40 μg/g (high-dose) PMO25 treatment increased exon 7 inclusion 2.5-fold

This dose-response relationship can be directly correlated with PMO25 tissue levels detected by the antibody.

How can PMO25 antibody be used to evaluate peptide-conjugated morpholino efficacy?

Peptide conjugation to PMO25 significantly enhances cellular uptake and therapeutic efficacy. Research on peptide-conjugated PMOs (like DG9-PMO) for exon skipping in Duchenne muscular dystrophy demonstrates improved therapeutic outcomes . PMO25 antibody enables researchers to:

• Compare distribution patterns: Immunohistochemical analysis can reveal differences in tissue penetration between unconjugated and peptide-conjugated PMO25

• Quantify cellular uptake efficiency: Flow cytometry with PMO25 antibody can measure the percentage of cells successfully transfected

• Assess clearance kinetics: Longitudinal sampling and PMO25 antibody detection can determine if peptide conjugation alters morpholino persistence in tissues

The advantages of peptide conjugation include:

• Enhanced cellular uptake

• Lower effective doses

• Improved tissue targeting

• Extended duration of effect

What molecular assays can complement PMO25 antibody detection to provide comprehensive therapeutic evaluation?

To thoroughly evaluate PMO25 efficacy, researchers should complement antibody detection with:

• RT-PCR for exon skipping quantification: Measuring the percentage of correctly skipped exons correlates with PMO25 distribution

• Protein restoration assessment: Western blot for target proteins (e.g., SMN in SMA models) confirms functional outcomes

• Histopathological evaluation: Assessing neuromuscular junction architecture, muscle fiber size, and motor neuron counts provides functional correlates

• Synapse quantification: Research shows that PMO25 treatment significantly affects vGLUT1-positive synapses on ChAT-positive motor neurons

In SMA mouse models, low-dose PMO25 treatment resulted in:

• 1.6-fold increase in SMN2 exon 7 inclusion

• Significant improvement in muscle fiber diameter compared to untreated type I SMA mice

• Intermediate endplate size changes between type I and type III mice

• Preservation of motor neurons and synaptic connections

What strategies can address poor signal-to-noise ratio when using PMO25 antibody?

When facing high background or weak specific signals with PMO25 antibody, researchers should consider:

• Optimize blocking conditions: Test different blocking agents (BSA, normal serum, commercial blockers) at various concentrations

• Adjust antibody concentration: Perform titration experiments to identify optimal primary antibody dilution

• Modify incubation parameters: Compare overnight incubation at 4°C versus shorter incubations at room temperature

• Enhance washing steps: Increase washing duration or frequency between antibody incubations

• Test signal amplification systems: For low-abundance targets, consider using tyramide signal amplification or other amplification methods

How can researchers validate PMO25 antibody specificity in their experimental system?

Proper validation of PMO25 antibody specificity is essential for reliable research outcomes and should include:

• Negative controls: Test antibody reactivity in tissues from untreated animals (no PMO25 exposure)

• Competitive inhibition: Pre-incubate antibody with purified PMO25 to demonstrate signal reduction

• Dose-dependent detection: Verify that signal intensity correlates with known PMO25 concentrations

• Comparison with orthogonal detection methods: Correlate antibody detection with mass spectrometry or other antibody-independent methods when possible

• Cross-reactivity assessment: Test reactivity against similar morpholino sequences to ensure specificity