NEDP1 Antibody

Basic Research Questions

How to validate NEDP1 antibody specificity in cellular models?

Validate using CRISPR/Cas9 knockout controls to confirm loss of signal in NEDP1-deficient cells . Combine with functional assays monitoring NEDD8 conjugate accumulation (e.g., western blot showing ~2.5-fold increase in NEDD8-modified proteins in knockout vs. wild-type cells) . Include orthogonal methods like immunoprecipitation-mass spectrometry to identify off-target interactions.

What experimental systems demonstrate NEDP1 antibody utility for substrate identification?

Use cytoplasmic fractionation protocols (e.g., differential centrifugation) since >90% of NEDP1 and its substrates localize to the cytoplasm . Pair with:

• PARP-1 NEDDylation assays (monitor K425/KQMR sites via mutagenesis)

• Sucrose gradient sedimentation for APAF1 oligomerization studies (Figure 5A in shows 80% reduction in APAF1 oligomers in NEDP1-/- cells post-DNA damage)

Advanced Mechanistic Questions

How to resolve contradictions in NEDP1's role in DNA damage response?

Address conflicting data through compartment-specific analysis:

What methodologies confirm NEDP1's role in ALS-related stress granule (SG) dynamics?

Implement:

• Live-cell imaging: Track SG dissolution kinetics (mutant TIA1 SGs dissolve 2.3x faster with NEDP1 inhibition )

• Phase separation assays: Quantify SG liquid-to-solid transition using FRAP (35% higher protein mobility in NEDP1-/- cells )

• Cross-species validation: Compare C. elegans motility (38% improvement in G85R SOD1 mutants ) with human neuron SG clearance rates

Technical Optimization Questions

How to design experiments distinguishing NEDP1 processing vs. deconjugation activity?

Employ sequential antibody-based approaches:

• Processing assay: Monitor pro-NEDD8 conversion using N-terminal specific antibodies (95% inhibition with 50nM Nb9 )

• Deconjugation assay: Measure cullin-NEDD8 levels via sandwich ELISA (IC50 <10nM for developed nanobodies )

• Temporal control: Inducible expression systems to separate early (0-6hr) processing effects from late (24hr+) deconjugation impacts

Translational Research Questions

What validation steps ensure NEDP1 antibody relevance for therapeutic development?

Establish three-tiered testing:

• Biochemical: Surface plasmon resonance (SPR) showing Kd <5nM

• Cellular: SG elimination assays in ALS patient fibroblasts (60% faster clearance vs controls )

• Organismal: C. elegans paralysis scoring (42% reduction in G4C2 repeat models )

How to address off-target effects in NEDP1 inhibition studies?

Implement:

• Proteome-wide NEDDylome analysis using wild-type NEDD8 (vs. R74K mutant)

• PARP-1 rescue experiments: Reintroduce WT vs K425R mutants in knockout background

• Dose-response curves comparing nanobody effects (e.g., Nb9 shows 100x selectivity over SENP2 )