ELMO1 Antibody, FITC conjugated

What cellular processes can be studied using FITC-conjugated ELMO1 antibodies?

FITC-conjugated ELMO1 antibodies are valuable tools for investigating several critical cellular processes, including:

• Cytoskeletal rearrangements during phagocytosis of apoptotic cells

• Cell migration and motility

• Interactions with DOCK1 and CRK in the formation of protein complexes

• Rac activation in various cell types

• Autophagy induction and bacterial clearance pathways

• Platelet function and thrombus formation

These antibodies allow for direct visualization of ELMO1 localization and expression patterns in different subcellular compartments without requiring secondary antibody detection steps .

What are the recommended applications for FITC-conjugated ELMO1 antibodies?

FITC-conjugated ELMO1 antibodies are particularly well-suited for:

• Immunofluorescence (IF) at dilutions of 1:50-200

• Immunohistochemistry on paraffin-embedded sections (IHC-P)

• Flow cytometry for detecting ELMO1 expression in specific cell populations

• Live cell imaging studies examining ELMO1 dynamics

The direct fluorophore conjugation eliminates potential cross-reactivity issues that can occur with secondary antibodies, making these particularly valuable for multicolor immunofluorescence studies .

What species reactivity should be considered when selecting ELMO1 antibodies?

When selecting ELMO1 antibodies, researchers should consider the following species reactivity patterns:

Always validate antibody reactivity in your specific experimental system, as cross-reactivity may vary between applications .

How should experiments be designed to study ELMO1's role in neutrophil migration using FITC-conjugated antibodies?

When designing experiments to study ELMO1's role in neutrophil migration, consider the following methodological approach:

• Model selection: Use both in vitro systems (isolated neutrophils) and in vivo models (such as zebrafish elmo1 mutants for live imaging)

• Baseline characterization: Establish normal migration parameters in wild-type cells using time-lapse microscopy

• ELMO1 manipulation: Compare ELMO1-deficient (elmo1-/-) neutrophils with wild-type controls

• Complementation studies: Express wild-type or mutant ELMO1 in ELMO1-deficient neutrophils to assess functional rescue

• Migration assays: Implement chemotaxis assays using inflammatory stimuli or bacterial infection models

• Visualization methodology: Use FITC-conjugated ELMO1 antibodies at 1:50-200 dilution for co-localization studies with cytoskeletal markers

This experimental framework has successfully demonstrated that ELMO1 deficiency significantly reduces neutrophil migration speed and their accumulation at inflammation sites .

What controls are necessary when using FITC-conjugated ELMO1 antibodies in flow cytometry?

When using FITC-conjugated ELMO1 antibodies in flow cytometry, the following controls are essential:

• Isotype control: Include a FITC-conjugated IgG of the same isotype (IgG1 kappa for mouse monoclonal or IgG for polyclonal) to assess non-specific binding

• Unstained control: Include cells without any antibody to establish baseline autofluorescence

• FMO control: Fluorescence Minus One control where all antibodies except ELMO1-FITC are included in a multicolor panel

• Positive control: Include a cell line known to express high levels of ELMO1 (H1299 cells or HeLa cells based on validation data)

• Negative control: If possible, include ELMO1 knockout cells (elmo1-/-)

• Titration experiment: Perform antibody titration (1:10, 1:50, 1:100, 1:200) to determine optimal signal-to-noise ratio

These controls are critical for distinguishing specific ELMO1 staining from background or non-specific fluorescence, particularly important when examining subtle changes in ELMO1 expression or localization .

How can researchers study ELMO1-DOCK1-Rac interactions using FITC-conjugated antibodies?

To investigate ELMO1-DOCK1-Rac interactions using FITC-conjugated ELMO1 antibodies, implement the following methodological approach:

• Co-immunoprecipitation followed by immunofluorescence:

  • Immunoprecipitate ELMO1 using non-conjugated antibodies

  • Probe for interaction partners (DOCK1, CRK, RhoG) by western blotting

  • Perform immunofluorescence with FITC-ELMO1 antibodies (1:500 dilution) and antibodies against interaction partners

• FRET biosensor approach:

  • Express RacFRET biosensor in cells of interest

  • Monitor FRET ratio changes to assess GTP-bound Rac activity

  • Compare FRET ratios between wild-type and ELMO1-deficient cells

  • Complement with FITC-ELMO1 antibody staining to correlate ELMO1 localization with Rac activation

• Active Rac1 pull-down assay:

  • Use GST-PBD to pull down GTP-bound active Rac1

  • Compare Rac1 activation between ELMO1-expressing and ELMO1-deficient cells

  • Combine with FITC-ELMO1 immunofluorescence to correlate protein localization with function

This combined approach has successfully demonstrated that ELMO1 deficiency results in reduced Rac binding to GTP, confirming ELMO1's role in Rac activation pathways .

What are the recommended fixation and permeabilization methods for optimal FITC-ELMO1 antibody staining?

For optimal FITC-ELMO1 antibody staining, consider these fixation and permeabilization methods based on application:

• For immunofluorescence in cultured cells:

  • Methanol fixation (shown effective for HeLa cells)

  • 10 minutes at -20°C for preservation of cytoskeletal structures

  • Alternative: 4% paraformaldehyde (10 minutes at room temperature) followed by 0.1% Triton X-100 permeabilization (5 minutes)

• For tissue sections:

  • Formalin-fixed paraffin-embedded (FFPE) sections

  • Heat-mediated antigen retrieval with sodium citrate buffer (pH 6.0)

  • 20 minutes boiling followed by 15-minute antibody incubation at room temperature

• For flow cytometry:

  • 2% paraformaldehyde (10 minutes at room temperature)

  • 0.1% saponin in PBS for permeabilization

  • Maintain 0.1% saponin in all washing and incubation steps

These methods have been validated in the literature, with methanol fixation specifically noted in product documentation for ELMO1 antibody ab155775 .

How can researchers troubleshoot non-specific binding when using FITC-conjugated ELMO1 antibodies?

When encountering non-specific binding with FITC-conjugated ELMO1 antibodies, implement this systematic troubleshooting approach:

• Increase blocking stringency:

  • Extend blocking time to 1-2 hours

  • Use 5-10% serum from the same species as secondary antibody

  • Include 0.1-0.3% Triton X-100 in blocking solution

  • Consider adding 1% BSA and 0.1% cold fish skin gelatin

• Optimize antibody concentration:

  • Perform a titration experiment (1:50, 1:100, 1:200, 1:500)

  • Select the dilution with highest specific signal and lowest background

• Modify washing protocols:

  • Increase number of washes (5-6 times)

  • Extend wash duration (10 minutes per wash)

  • Add 0.05% Tween-20 to wash buffers

• Include validated controls:

  • ELMO1 knockout or knockdown samples

  • Peptide competition assay using the immunizing peptide

• Consider autofluorescence reduction:

  • Treat samples with 0.1% Sudan Black B in 70% ethanol

  • Alternatively, use commercial autofluorescence reducers

These steps have helped researchers achieve specific ELMO1 staining in challenging tissue samples like human lymphoid tissue and neuronal tissue .

How should researchers interpret changes in ELMO1 localization observed with FITC-conjugated antibodies?

When interpreting ELMO1 localization changes visualized with FITC-conjugated antibodies, consider:

• Baseline cellular distribution:

  • ELMO1 is primarily cytoplasmic under resting conditions

  • Observe for punctate structures that may represent intracellular vesicles

• Translocation patterns:

  • Membrane recruitment during phagocytosis or cell migration

  • Co-localization with DOCK1/2 at membrane protrusions

  • Association with autophagosomal structures marked by LC3B after bacterial infection

• Context-dependent interpretation:

  • In neutrophils: Translocation to the leading edge during chemotaxis

  • In macrophages: Association with phagocytic cups and intracellular vesicles

  • In platelets: Distribution changes during spreading and activation

• Quantitative assessment:

  • Measure intensity ratios between different cellular compartments

  • Track temporal changes in localization following stimulation

  • Correlate localization changes with functional outcomes

Studies have demonstrated that ELMO1 can be found in intracellular vesicles and exhibits enhanced accumulation of LC3B following engulfment of bacteria or treatment with autophagy-inducing rapamycin, indicating its role in autophagy pathways .

How can FITC-conjugated ELMO1 antibodies be used to study ELMO1's role in autophagy?

To investigate ELMO1's role in autophagy using FITC-conjugated antibodies, implement this methodological approach:

• Co-localization studies:

  • Use FITC-ELMO1 antibodies (1:100 dilution) together with markers for:

    • LC3B (autophagosome marker)

    • ATG5 (early autophagy protein)

    • ULK1 (autophagy initiation kinase)

  • Quantify co-localization under basal conditions and after autophagy induction

• Autophagic flux assessment:

  • Treat cells with bafilomycin A1 to block lysosomal degradation

  • Monitor ELMO1 association with accumulated autophagosomes

  • Compare ELMO1-autophagosome association in control vs. autophagy-deficient cells

• LC3-associated phagocytosis (LAP) assay:

  • Expose cells to phagocytic stimuli (bacteria, zymosan)

  • Visualize ELMO1 localization to LC3+ phagosomes

  • Compare LAP efficiency between wild-type and ELMO1-deficient cells

• pH and proteolytic activity assessment:

  • Use pH-sensitive dyes to monitor phagosomal acidification

  • Employ DQ-BSA beads to assess proteolytic activity in phagosomes

  • Compare between control and ELMO1-depleted cells

Research has demonstrated that ELMO1 regulates LC3B accumulation through ATG5-dependent but ULK1-independent mechanisms, suggesting preferential involvement in LC3-associated phagocytosis rather than classical autophagy .

What are the methodological considerations when studying ELMO1 variants using antibody-based approaches?

When studying ELMO1 variants using antibody-based approaches, researchers should consider these methodological aspects:

• Epitope accessibility:

  • Check if the variant affects the antibody binding site

  • For C-terminal antibodies (like ab155775), variants affecting C-terminal structure may reduce detection

  • Use multiple antibodies targeting different epitopes for verification

• Expression system selection:

  • Consider transient expression in relevant cell types

  • Zebrafish elmo1 mutants provide an excellent in vivo system for functional verification

  • Ensure physiological expression levels to avoid artifacts

• Functional domain analysis:

  • Use FITC-ELMO1 antibodies to assess localization of variants affecting:

    • ELMO Inhibitory Domain (EID)

    • PH domain (interacts with DOCK or RAC)

    • RhoG binding regions

  • Compare wild-type vs. variant localization patterns

• Controls for variant studies:

  • Include known functional variants (p.E90K, p.D194G)

  • Include known loss-of-function variants (p.R354X)

  • Perform functional rescue experiments in ELMO1-deficient backgrounds

Functional verification of human ELMO1 variants using zebrafish neutrophils has demonstrated that variants p.E90K and p.D194G maintain functional activity, while the p.R354X variant fails to rescue the migration defects in ELMO1-deficient cells .

How can FITC-conjugated ELMO1 antibodies be used to study ELMO1's role in autoimmune diseases?

For investigating ELMO1's role in autoimmune diseases using FITC-conjugated antibodies, implement this research strategy:

• Tissue-specific expression analysis:

  • Compare ELMO1 expression in tissues from patients with autoimmune diseases vs. healthy controls

  • Use FITC-ELMO1 antibodies (1:50-200 dilution) for immunohistochemistry on patient samples

  • Quantify expression differences and correlate with disease severity

• Immune cell phenotyping:

  • Perform flow cytometry using FITC-ELMO1 antibodies on:

    • Neutrophils (key ELMO1-expressing cells in rheumatoid arthritis)

    • Macrophages/monocytes

    • T and B lymphocytes

  • Compare expression patterns between patient and control immune cells

• Functional studies in disease models:

  • Use ELMO1-FITC to track neutrophil behavior in arthritis models

  • Correlate ELMO1 expression with neutrophil accumulation at inflammatory sites

  • Assess the impact of disease-associated ELMO1 variants on protein localization

• Single-cell analysis approach:

  • Combine FITC-ELMO1 antibody staining with single-cell RNA sequencing

  • Identify cell populations with altered ELMO1 expression in disease states

  • Correlate with expression of other disease-relevant genes

Research has identified single nucleotide polymorphisms (SNPs) in the ELMO1 gene associated with rheumatoid arthritis, with ELMO1 deficiency in neutrophils specifically protecting against inflammatory arthritis in mouse models .

What methodological approaches can be used to study ELMO1's role in platelet function?

To investigate ELMO1's role in platelet function using FITC-conjugated antibodies, implement the following methodology:

• Platelet isolation and activation studies:

  • Isolate platelets from wild-type and ELMO1-deficient models

  • Activate with various agonists (GPVI agonists, PAR4 agonists)

  • Use FITC-ELMO1 antibodies to track protein localization during activation

  • Correlate with functional readouts (aggregation, secretion, spreading)

• Integrative functional assessment:

  • Analyze platelet aggregation in response to various agonists

  • Measure granule secretion and integrin αIIbβ3 activation

  • Quantify thromboxane generation

  • Compare results between wild-type and ELMO1-deficient platelets

• Spreading dynamics visualization:

  • Perform real-time imaging of platelets spreading on immobilized fibrinogen

  • Track ELMO1-FITC localization during spreading

  • Quantify spreading kinetics and correlate with ELMO1 distribution

• RhoG activity correlation:

  • Measure RhoG activation in platelets following GPVI stimulation

  • Compare RhoG activity between wild-type and ELMO1-deficient platelets

  • Correlate with ELMO1 localization patterns

Research has demonstrated that ELMO1 deficiency enhances platelet function, with ELMO1-/- platelets showing enhanced aggregation, granule secretion, integrin activation, and thromboxane generation in response to GPVI agonists, suggesting ELMO1 negatively regulates GPVI-mediated thrombus formation via RhoG .