ELP1 Antibody, FITC conjugated

What is ELP1 and why is it significant in research?

ELP1 (Elongator Complex Protein 1), also known as IKAP (IκB kinase complex-associated protein) or p150, functions as a scaffolding subunit of the six-member Elongator complex. This complex plays a critical role in modifying particular tRNAs, especially wobble uridines in the anti-codon sequence . ELP1 has gained significant research interest due to its mutation being causative in Familial Dysautonomia (FD), a rare sensory and autonomic neuropathy . Research using this protein focuses on neuronal development, DNA repair mechanisms, and cellular signaling pathways, making ELP1 antibodies valuable tools for investigating these biological processes.

What are the key applications for FITC-conjugated ELP1 antibodies?

FITC-conjugated ELP1 antibodies are primarily utilized in fluorescence-based applications including:

• Immunofluorescence analysis of cultured cells (IF-cc) and paraffin-embedded tissue sections (IF-p)

• Flow cytometry for detecting ELP1 expression in cell populations

• Confocal microscopy for subcellular localization studies

• Multiplex immunofluorescence when combined with other differently-labeled antibodies

The FITC conjugation provides a strong green fluorescence signal (excitation ~495 nm, emission ~520 nm) that is compatible with standard fluorescence microscopy filter sets and flow cytometers.

How do researchers select the appropriate ELP1 antibody based on target epitopes?

Selection should be based on experimental goals and target regions of interest:

When studying neuronal phenotypes in mouse models of Familial Dysautonomia, antibodies reactive to mouse/rat ELP1 (AA 1151-1250) are most appropriate .

What is the optimal protocol for immunofluorescence using FITC-conjugated ELP1 antibody?

Standard Protocol for Cell Cultures:

• Fix cells with 4% paraformaldehyde (10 minutes at room temperature)

• Permeabilize with 0.1% Triton X-100 in PBS (5 minutes)

• Block with 5% normal serum in PBS (1 hour)

• Incubate with FITC-conjugated ELP1 antibody (dilution 1:50-1:200 determined through titration) overnight at 4°C

• Wash 3× with PBS

• Counterstain nucleus with DAPI

• Mount with anti-fade mounting medium

Optimization Notes:

• For neuronal tissues, extend fixation to 15-20 minutes

• Use lower antibody concentrations (1:200-1:500) for tissues with high ELP1 expression

• Include a no-primary antibody control to assess background autofluorescence

How can researchers validate the specificity of ELP1 antibodies?

Methodological approach to antibody validation:

• Genetic controls: Compare staining between wild-type and Elp1-deficient mouse embryonic fibroblasts (MEFs)

• Western blot verification: Confirm single band at expected molecular weight (~150 kDa)

• Peptide competition: Pre-incubate antibody with immunizing peptide (AA 1107-1258 or AA 1151-1250) before staining

• Cross-validation: Compare staining patterns using multiple antibodies targeting different ELP1 epitopes

• siRNA knockdown: Reduced signal following ELP1 siRNA treatment confirms specificity

What sample preparation methods optimize ELP1 detection in tissues?

For optimal ELP1 detection in tissue sections:

• Use fresh-frozen or lightly fixed tissue (overfixation can mask epitopes)

• For paraffin sections, perform heat-mediated antigen retrieval (citrate buffer pH 6.0)

• Extend blocking time to 2 hours to minimize background in neural tissues

• Include 0.1% Tween-20 in wash buffers to reduce non-specific binding

• For enteric nervous system studies, use thick sections (25-30 μm) to capture neuronal networks

How can FITC-conjugated ELP1 antibodies be used to study neuronal development in Familial Dysautonomia models?

Methodological approach for studying FD using ELP1 antibodies:

• Neuronal culture analysis: Compare ELP1 localization in differentiated patient-derived iPSCs versus controls

• Ganglion development: Track ELP1 expression in cranial, dorsal root, and sympathetic ganglia during development in mouse models

• Enteric nervous system: Use FITC-ELP1 antibodies in combination with neuronal markers (PGP9.5, βIII-tubulin) to assess enteric plexus formation in Tuba1a-Cre;Elp1^loxp/loxp^ conditional knockout mice

• Quantitative analysis: Measure neurite outgrowth and branching in ELP1-labeled neurons

Research findings show that conditional deletion of Elp1 in neurons disrupts enteric nervous system development and alters gut epithelial integrity, particularly affecting E-cadherin expression at adherens junctions .

What approaches can researchers use to study ELP1's role in DNA repair mechanisms?

Based on recent research findings:

• DNA damage assays: Conduct comet assays in Elp1-deficient MEFs to assess double-strand break (DSB) formation following irradiation

• RAD51 co-localization: Perform dual immunofluorescence with FITC-ELP1 and RAD51 antibodies to study homologous recombination repair foci

• Rescue experiments: Transfect Elp1-deficient cells with hRAD51-expressing vectors and assess repair efficiency

• Chromosome spreading: Analyze metaphase chromosome abnormalities in Elp1-deficient cells following DNA damage

Research has revealed that ELP1 facilitates RAD51-mediated homologous recombination repair, linking translational regulation to DNA repair processes .

How can ELP1 antibodies be utilized to investigate therapeutic applications in cancer research?

Emerging research has employed ELP1-based constructs in therapeutic applications:

• Thermal targeting strategies: Study the cellular uptake of SynB1-ELP1-p50 constructs at different temperatures using confocal microscopy

• Apoptosis induction: Use FITC-annexin V in combination with ELP1 antibodies to assess apoptotic induction in cancer cells treated with ELP1-peptide conjugates

• Subcellular localization: Track the internalization of rhodamine-conjugated SynB1-ELP1-p50 at 37°C versus 42°C to demonstrate thermally-enhanced uptake

• Therapeutic response: Monitor changes in NFκB signaling following treatment with ELP1-conjugated inhibitory peptides

Research demonstrates that SynB1-ELP1-p50 induces apoptosis and reduces proliferation in MDA-MB-231 breast cancer cells, with enhanced effects when combined with hyperthermia .

What are common issues when working with FITC-conjugated antibodies and how can they be resolved?

How should researchers interpret ELP1 localization patterns in different cell types?

ELP1 typically shows both cytoplasmic and nuclear localization, with variations depending on cell type and physiological state:

• Neurons: Predominantly cytoplasmic with enrichment in growth cones during development

• Fibroblasts: Diffuse cytoplasmic pattern with nuclear exclusion

• Cancer cells: May show altered localization with increased nuclear presence

Quantitative analysis should include:

• Cytoplasmic-to-nuclear ratio measurements

• Co-localization coefficients with organelle markers

• Intensity profile analysis across cellular compartments

What controls are essential when studying ELP1 expression in disease models?

Essential controls include:

• Positive control: Known ELP1-expressing cell line (e.g., fibroblasts) to confirm antibody functionality

• Negative control: Elp1-deficient cells or tissues where available

• Isotype control: Rabbit IgG-FITC at matching concentration to assess non-specific binding

• Autofluorescence control: Unstained sample to establish background fluorescence levels

• Absorption control: Pre-incubate antibody with immunizing peptide to confirm specificity

How might researchers integrate ELP1 antibodies with emerging imaging technologies?

Methodological innovations for future research:

• Super-resolution microscopy: Use photoconvertible fluorophore-conjugated ELP1 antibodies for PALM/STORM imaging of Elongator complex organization

• Live-cell imaging: Develop cell-permeable nanobodies against ELP1 for tracking dynamics in living neurons

• Tissue clearing techniques: Combine FITC-ELP1 antibodies with CLARITY or iDISCO for whole-organ imaging of enteric nervous system in FD models

• Multiplexed imaging: Implement cyclic immunofluorescence or mass cytometry to simultaneously analyze ELP1 with dozens of other proteins in tissue sections

What are promising approaches for studying ELP1 in the context of RNA modification?

Since ELP1 functions within the Elongator complex to modify tRNAs:

• tRNA modification analysis: Couple ELP1 immunoprecipitation with mass spectrometry to identify associated tRNAs

• Translation efficiency assays: Correlate ELP1 levels with polysome profiling data to assess impacts on translation

• RNA-protein interaction studies: Use CLIP-seq methodologies with ELP1 antibodies to map binding sites

• Single-molecule approaches: Implement super-resolution imaging to visualize ELP1-tRNA interactions in situ