ELMO1 Antibody, Biotin conjugated

What is ELMO1 and what are its primary biological functions?

ELMO1 (Engulfment and Cell Motility Protein 1) plays a critical role in cytoskeletal rearrangements essential for phagocytosis of apoptotic cells and cellular motility. It functions in association with DOCK1 and CRK proteins, initially proposed as necessary in complex with DOCK1 to activate Rac Rho small GTPases. Current research indicates ELMO1 may enhance the guanine nucleotide exchange factor (GEF) activity of DOCK1 . This 84 kDa protein is fundamental to immune cell function, particularly in processes involving cellular engulfment mechanisms and inflammatory responses .

ELMO1 has been implicated in inflammatory bowel disease (IBD) pathophysiology, where it participates in bacterial internalization in gut epithelial cells. Elevated ELMO1 expression correlates positively with pro-inflammatory cytokines like TNF-α and MCP-1, with approximately 4-fold higher expression observed in patients with active Crohn's disease compared to healthy controls . This suggests ELMO1 plays a significant role in regulating inflammatory responses in the intestinal epithelium.

What experimental applications are suitable for biotin-conjugated ELMO1 antibodies?

Biotin-conjugated ELMO1 antibodies offer versatility across multiple experimental platforms. The conjugation-ready formats are specifically designed for advanced applications including:

• Flow cytometry (particularly intracellular detection)

• Immunocytochemistry/Immunofluorescence (ICC/IF)

• Multiplex imaging applications

• ELISA-based detection systems

• Mass cytometry

• Immunoprecipitation (IP) experiments

The biotinylated ELMO1 antibody is particularly valuable in sandwich ELISA techniques, where it functions as the detection antibody following primary capture. This configuration allows for highly sensitive and specific quantification of ELMO1 in biological samples including serum, plasma, and cell culture supernatants . The high affinity between biotin and streptavidin enables signal amplification through subsequent addition of streptavidin-HRP conjugates.

How should biotin-conjugated ELMO1 antibodies be stored and handled to maintain optimal activity?

For maximum stability and preserved immunoreactivity, biotin-conjugated ELMO1 antibodies should be stored at 2-8°C and used within 12 months of the manufacturing date . Before experimental use, all reagents should be equilibrated to room temperature (18-25°C). Following experimental procedures, antibodies should be returned promptly to appropriate storage conditions to maintain functionality .

The biotin conjugate is sensitive to repeated freeze-thaw cycles, which should be minimized. Aliquoting the antibody upon first thaw is recommended for long-term studies. Additionally, because biotin-conjugated antibodies can be light-sensitive, particularly when combined with enzyme substrates like TMB, storage in amber vials or protection from direct light exposure during experimental protocols is advisable .

What is the optimal approach for validating specificity of biotin-conjugated ELMO1 antibodies?

Validating antibody specificity requires a multi-dimensional approach:

• Western blot validation: The predicted molecular weight for human ELMO1 is 84 kDa. Validation should include human cell lines known to express ELMO1, such as HeLa or H1299 cells. For example, research has demonstrated successful detection in H1299 whole cell lysate (30 μg) using a 12% SDS-PAGE system .

• Positive and negative control tissues/cells: Include samples with known ELMO1 expression profiles. IHC studies have shown variable ELMO1 expression in normal gut epithelium and lamina propria, with significantly elevated expression in inflamed epithelium from IBD patients .

• Peptide competition assay: Pre-incubation with the immunizing peptide (particularly relevant for antibodies raised against the C-terminal region, aa 650-C-terminus or aa 700-C-terminus) should abolish specific signal.

• Cross-reactivity assessment: When working with human ELMO1, testing cross-reactivity with mouse and rat samples can provide additional validation metrics, as several antibodies demonstrate cross-species reactivity due to sequence homology .

• Signal-to-noise ratio optimization: When using biotin-conjugated antibodies in complex samples, careful titration is essential to determine the optimal antibody concentration that maximizes specific signal while minimizing background.

What methodological approaches enable quantitative assessment of ELMO1 in clinical samples?

Quantitative assessment of ELMO1 in clinical samples can be achieved through several complementary techniques:

• Sandwich ELISA methodology: The most precise quantification utilizes a sandwich ELISA system where:

  • Capture antibody (anti-ELMO1 monoclonal) is pre-coated on microwells

  • Sample containing ELMO1 is added and captured

  • Biotinylated detection antibody binds to captured ELMO1

  • Streptavidin-HRP conjugate forms complex with biotinylated antibody

  • TMB substrate enables colorimetric detection at 450nm

  This approach provides sensitivity in the picogram/ml range, with standard curves typically ranging from 31.25-2000 pg/ml using an initial 4000 pg/ml standard with serial dilution .

• Immunohistochemical quantification: For tissue biopsies, immunohistochemistry enables spatial assessment of ELMO1 expression patterns. Studies demonstrate that in IBD patients, ELMO1 expression is elevated in both epithelium and lamina propria, with the most dramatic increases observed in diseased epithelium . Quantification should include:

  • Multiple fields per sample (minimum 5)

  • Assessment of both epithelial and lamina propria compartments

  • Scoring systems calibrated to cellular intensity and percentage positivity

• mRNA-protein correlation: For comprehensive analysis, correlating protein levels with mRNA expression provides deeper mechanistic insights. RT-qPCR analysis has demonstrated approximately 4-fold elevation in ELMO1 mRNA in active Crohn's disease patients compared to healthy controls .

What experimental design considerations are important when studying ELMO1-mediated bacterial internalization?

When designing experiments to study ELMO1-mediated bacterial internalization, researchers should consider:

• Cell model selection:

  • Primary enteroids derived from stem cells provide a physiologically relevant model system for studying epithelial ELMO1 function

  • Cell lines should be selected based on endogenous ELMO1 expression levels

  • ELMO1 knockout or knockdown controls are essential for validation

• Bacterial strain considerations:

  • Adherent-invasive E. coli strains (e.g., AIEC-LF82) associated with Crohn's disease represent clinically relevant models

  • Fluorescently-labeled bacteria enable visualization and quantification of internalization events

  • MOI (multiplicity of infection) should be carefully titrated for each experimental system

• Quantification methodology:

  • Flow cytometry enables high-throughput assessment of bacterial internalization

  • Confocal microscopy permits visualization of localization with cytoskeletal elements

  • Gentamicin protection assays differentiate adherent from internalized bacteria

• Downstream inflammatory readouts:

  • MCP-1 production follows ELMO1-dependent bacterial internalization

  • TNF-α serves as a secondary inflammatory marker

  • Cytokine ELISAs provide quantitative assessment of these inflammatory mediators

Research has demonstrated that ELMO1 facilitates bacterial entry into epithelial cells through tight junctions, triggering subsequent MCP-1 production. This ELMO1-MCP-1 axis then recruits monocytes to sites of inflammation, where bacteria can enter monocytes in an ELMO1-dependent manner, perpetuating inflammatory cascades through TNF-α release .

How can biotin-conjugated ELMO1 antibodies be optimized for multiplex imaging applications?

Optimizing biotin-conjugated ELMO1 antibodies for multiplex imaging requires systematic approach:

• Sequential detection strategy:

  • Initial staining with non-biotinylated primary antibodies against other targets

  • Secondary staining with fluorophore-conjugated secondary antibodies

  • Biotin-blocking step to neutralize endogenous biotin

  • Addition of biotin-conjugated ELMO1 antibody

  • Detection with streptavidin-conjugated fluorophore distinct from previous channels

• Titration matrix optimization:

  • Determine optimal antibody concentration range (typically starting at 1:500 dilution for immunofluorescence)

  • Assess performance across concentration gradient

  • Evaluate signal-to-background ratio at each concentration

  • Select concentration that maximizes specific signal while minimizing background

• Fixation methodology considerations:

  • Methanol fixation has been validated for ELMO1 detection in HeLa cells using immunofluorescence

  • Paraformaldehyde fixation with appropriate permeabilization may be required for multiplexing with certain epitopes

  • Epitope retrieval methods should be compatible across all target proteins

• Counterstaining strategy:

  • Nuclear counterstain (e.g., Hoechst 33342) provides spatial context

  • Additional markers for cellular compartments help delineate ELMO1 localization

  • Z-stack acquisition may be necessary to fully capture ELMO1 distribution

• Spectral unmixing:

  • In cases where fluorophore emission spectra overlap, spectral unmixing algorithms can separate signals

  • Single-stained controls are essential for accurate unmixing

What is the relationship between ELMO1 expression and inflammatory signaling in disease models?

The relationship between ELMO1 expression and inflammatory signaling reveals complex regulatory mechanisms:

• Boolean relationship with inflammatory mediators:

  • RNA-Seq analysis of 214 normal colon samples revealed a Boolean relationship between ELMO1 and MCP-1 expression, where high levels of one typically correspond with elevated levels of the other

  • This pattern suggests a fundamental gene expression signature conserved despite population variance

• Quantitative correlation with disease activity:

  • ELMO1 expression is elevated approximately 4-fold in active Crohn's disease compared to healthy controls

  • TNF-α and MCP-1 show concurrent 6-fold elevation in the same patients

  • This correlation suggests coordinated regulation of these inflammatory mediators

• Cell-specific expression patterns:

  • ELMO1 expression is detected in both epithelium and lamina propria in normal gut

  • In inflammatory bowel disease, expression is dramatically increased in the epithelium

  • This differential upregulation suggests cell-type specific regulatory mechanisms

• Functional consequences of elevated expression:

  • ELMO1 facilitates bacterial entry into epithelial cells

  • This entry triggers MCP-1 production

  • MCP-1 recruits monocytes to sites of inflammation

  • In monocytes, ELMO1-dependent bacterial entry triggers TNF-α release

  • This creates a feed-forward inflammatory loop that perpetuates chronic inflammation

This relationship suggests ELMO1 represents a potential therapeutic target that could simultaneously interrupt both the ELMO1-MCP-1 axis in epithelial cells and the ELMO1-TNF-α axis in macrophages, potentially addressing multiple inflammatory pathways in IBD.

What statistical approaches are most appropriate for analyzing ELMO1 expression data in clinical samples?

When analyzing ELMO1 expression data from clinical samples, these statistical approaches offer robust analysis:

• Two-tailed Student's t-test:

  • Appropriate for comparing ELMO1 expression between two defined groups (e.g., healthy vs. disease)

  • Has been successfully applied in analyzing bacterial internalization, monocyte recruitment, and ELISA results related to ELMO1 function

  • Results should be expressed as mean ± standard deviation

  • Statistical significance threshold typically set at p < 0.05

• Correlation analysis for expression patterns:

  • Pearson or Spearman correlation coefficients assess relationships between ELMO1 and inflammatory markers

  • Boolean relationship analysis can identify binary expression patterns within population datasets

  • Correlation matrices can visualize relationships across multiple inflammatory markers simultaneously

• Multivariate analysis for heterogeneous populations:

  • Principal component analysis (PCA) can identify patterns in high-dimensional datasets

  • Hierarchical clustering can group patients based on expression profiles

  • These approaches are particularly valuable when analyzing heterogeneous expression patterns observed in human populations

• Power analysis for study design:

  • Given the heterogeneous expression of ELMO1 in healthy humans (approximately 0.10 Arbitrary Units with substantial variance), power analysis should guide sample size determination

  • Observed differences between healthy and IBD populations (p = 0.036) suggest moderate effect sizes requiring adequate sampling

• Normalized expression metrics:

  • For RNA quantification, normalization to housekeeping genes is essential

  • For protein quantification, normalization to total protein or housekeeping proteins provides comparable metrics

  • Fold-change relative to control samples offers interpretable measurement units

How can non-specific binding be minimized when using biotin-conjugated ELMO1 antibodies?

Non-specific binding with biotin-conjugated antibodies presents unique challenges requiring systematic troubleshooting:

• Endogenous biotin blocking:

  • Tissues and cells contain endogenous biotin that can cause background

  • Pre-blocking with avidin/biotin blocking kits is essential

  • Sequential application of avidin followed by biotin saturates endogenous biotin and blocking reagent

• Optimal buffer composition:

  • For Western blotting, 12% SDS-PAGE systems have been validated for ELMO1 detection

  • For immunofluorescence, methanol fixation protocols yield specific signal at 1:500 antibody dilution

  • For ELISA applications, specialized diluents for standards and samples minimize matrix effects

• Cross-adsorption strategies:

  • If working with multiple species, cross-adsorbed detection reagents minimize species cross-reactivity

  • Secondary reagents should be carefully selected to avoid binding to endogenous immunoglobulins

• Titration optimization:

  • Systematic antibody dilution series identifies optimal concentration

  • Signal-to-noise ratio assessment at each concentration guides selection

  • For multiplex applications, individual optimization of each antibody prevents channel bleed-through

• Sample-specific considerations:

  • Serum and plasma samples may require additional blocking steps to minimize non-specific protein binding

  • Cell culture supernatants typically require less extensive blocking

  • Tissue sections benefit from hydrogen peroxide treatment to quench endogenous peroxidase activity when using HRP detection systems

What are the most effective approaches for detecting low-abundance ELMO1 in experimental samples?

Detecting low-abundance ELMO1 requires signal amplification strategies:

• Enhanced detection systems for immunoblotting:

  • Chemiluminescent substrates with extended signal duration

  • Longer exposure times with low-noise detection systems

  • Signal enhancers that amplify HRP activity

  • Loading higher protein concentrations (validated with 30 μg of whole cell lysate)

• Sensitivity optimization for ELISA:

  • Sandwich ELISA format with optimized antibody pairs

  • Extended sample incubation times at 4°C

  • Signal amplification with polymer-HRP systems

  • Enhanced substrate development techniques

  • Standard curves beginning at 31.25 pg/ml enable quantification of low abundance samples

• Enrichment techniques:

  • Immunoprecipitation to concentrate ELMO1 prior to analysis

  • Cell fractionation to isolate compartments with higher ELMO1 concentration

  • Targeted cell isolation from heterogeneous samples based on known expression patterns

• Tyramide signal amplification (TSA):

  • For immunohistochemistry and immunofluorescence applications

  • Provides 10-100 fold signal enhancement

  • Particularly valuable for detecting ELMO1 in normal gut tissue where expression is heterogeneous

• qPCR as complementary approach:

  • When protein detection reaches sensitivity limits, mRNA quantification provides complementary data

  • Digital PCR offers absolute quantification for low-copy transcripts

  • Correlation between mRNA and protein levels should be established for interpretation

How can researchers troubleshoot discrepancies between ELMO1 protein and mRNA expression levels?

Discrepancies between ELMO1 protein and mRNA levels require systematic investigation:

• Post-transcriptional regulation assessment:

  • MicroRNA regulation may cause protein-mRNA discordance

  • RNA-binding protein influence on translation efficiency

  • Analyze 3'UTR for regulatory elements affecting translation

• Protein stability considerations:

  • Protein half-life may differ between experimental conditions

  • Proteasomal degradation pathways may be differentially active

  • Proteasome inhibitors can help determine if protein stability contributes to observed discrepancies

• Antibody epitope accessibility:

  • ELMO1 antibodies target different regions (e.g., C-terminal regions aa 650-C-terminus or aa 700-C-terminus)

  • Protein-protein interactions or conformational changes may mask epitopes

  • Multiple antibodies targeting different epitopes can resolve this issue

• Isoform-specific detection:

  • Alternatively spliced isoforms may not be detected by all antibodies

  • PCR primers may amplify multiple transcript variants

  • Isoform-specific primers and antibodies provide clarification

• Subcellular localization changes:

  • ELMO1 distribution between cellular compartments may change under different conditions

  • Total protein extraction versus compartment-specific extraction yields different results

  • Fractionation studies followed by immunoblotting of individual fractions can resolve apparent discrepancies