ELMO1 Antibody, HRP conjugated

What is ELMO1 and what biological functions does it serve?

ELMO1 is a cytoplasmic protein involved in multiple critical cellular processes. It functions primarily in cytoskeletal rearrangements required for phagocytosis of apoptotic cells and cell motility. ELMO1 acts in association with DOCK1 and CRK proteins to enhance the guanine nucleotide exchange factor (GEF) activity of DOCK1, which is crucial for activating Rac Rho small GTPases .

Recent research has identified additional non-canonical roles for ELMO1, particularly as a cytoplasmic regulator of specific neutrophil receptors and as a promoter of arthritis . Furthermore, ELMO1 plays a protective role in renal structure maintenance and glomerular function, especially under diabetic conditions, by decreasing apoptosis in renal cells .

What applications is ELMO1 Antibody, HRP conjugated suitable for?

ELMO1 Antibody, HRP conjugated is primarily designed for Western blot (WB) applications . The HRP conjugation eliminates the need for secondary antibody incubation, streamlining the experimental workflow and potentially reducing background signal. The recombinant monoclonal variants offer high specificity and reproducibility for detection of human ELMO1 . While Western blot is the validated application, some ELMO1 antibodies (not necessarily HRP-conjugated versions) are suitable for immunohistochemistry on paraffin-embedded sections (IHC-P) and immunocytochemistry/immunofluorescence (ICC/IF) .

What are the optimal dilution ranges for ELMO1 Antibody, HRP conjugated?

For Western blot applications, the recommended dilution range for ELMO1 Antibody, HRP conjugated varies by manufacturer and specific antibody:

• The bsm-61890r-hrp antibody has a recommended dilution range of 1:300-5000 for Western blot

• The ab204386 (EPR12919) antibody performs optimally at a 1:5000 dilution for Western blot

Optimal dilution should be determined empirically for each experimental setup, considering factors such as protein expression levels, sample type, and detection method. When using new lots or with different sample types, titration experiments are advisable to determine the optimal working concentration.

What are the proper storage conditions for ELMO1 Antibody, HRP conjugated?

ELMO1 Antibody, HRP conjugated should be stored at -20°C according to manufacturer recommendations . To maintain antibody activity, it's recommended to aliquot the antibody into multiple vials upon receipt to avoid repeated freeze-thaw cycles, which can degrade the HRP enzyme and reduce signal strength. The antibody is typically supplied in a storage buffer containing glycerol (often 50%) and stabilizers such as BSA (1%) and preservatives like Proclin300 (0.02%) .

What is the cellular localization of ELMO1 protein?

ELMO1 is primarily localized in the cytoplasm and at the cell membrane . Immunofluorescence studies show a diffuse cytoplasmic distribution pattern . In specialized contexts, ELMO1 can translocate to specific subcellular regions during processes like phagocytosis or cell migration, where it participates in cytoskeletal rearrangements. In kidney tissue, ELMO1 has been localized in glomerular podocytes and tubules through immunohistochemical staining .

How can ELMO1 Antibody, HRP conjugated be used to study ELMO1's role in bacterial internalization?

ELMO1 plays a critical role in bacterial internalization by phagocytes. For studying this process, researchers can design experiments using the following approach:

• Cell preparation: Use macrophage cell lines (like J774) or primary macrophages from control and ELMO1-depleted sources (using shRNA knockdown or ELMO1-deficient mice).

• Bacterial infection model: Infect cells with appropriate bacterial strains (e.g., Salmonella) at controlled multiplicities of infection (MOI).

• Detection of internalization: After infection, lyse cells and plate lysates on bacterial growth media to quantify intracellular bacteria. Alternatively, use fluorescently labeled bacteria and flow cytometry for quantification.

• Western blot analysis: Use ELMO1 Antibody, HRP conjugated (1:5000 dilution) to confirm ELMO1 expression levels in control versus ELMO1-depleted cells.

• Downstream signaling: Assess activation of NFκB, ERK1/2, and p38 MAPK pathways, which are impaired in ELMO1-depleted cells following bacterial challenge .

Research has shown that ELMO1-depleted macrophages show significantly reduced bacterial internalization and subsequent cytokine production. ELMO1 is indispensable for bacterial internalization by intestinal macrophages, making this an important area for research into innate immunity .

What experimental design is recommended for studying ELMO1-mediated Rac1 activation?

ELMO1 plays a crucial role in activating Rac1, a process essential for phagocytosis and cytoskeletal rearrangements. To investigate this relationship using ELMO1 Antibody, HRP conjugated:

• Pull-down assay setup:

  • Prepare control and ELMO1-depleted cells (through knockdown or knockout)

  • Stimulate cells with appropriate stimuli (e.g., bacterial infection)

  • At different time points (e.g., 15 min, 30 min, 1 hour), perform Rac1-GTP pull-down assays

• Western blot analysis:

  • Load equal amounts of pull-down products and total cell lysates

  • Use ELMO1 Antibody, HRP conjugated to confirm ELMO1 expression levels

  • Use Rac1-specific antibody to detect active (GTP-bound) Rac1

• Quantification:

  • Normalize active Rac1 to total Rac1 expression

  • Compare activation kinetics between control and ELMO1-depleted cells

Studies have shown that ELMO1 depletion significantly impairs Rac1 activation after stimulation, with up to 6-fold less Rac1 activity in ELMO1-depleted cells compared to controls at peak activation times .

How can researchers investigate ELMO1's role in inflammatory cytokine production?

ELMO1 plays a significant role in regulating inflammatory cytokine production, particularly in response to bacterial infection. To study this function:

• Cell preparation:

  • Use control and ELMO1-depleted macrophages (through shRNA or CRISPR)

  • Consider both cell lines and primary cells from ELMO1-deficient models

• Stimulation protocol:

  • Challenge cells with appropriate stimuli (bacterial infection, LPS, etc.)

  • Include conditions with Rac1 inhibitors to dissect ELMO1-Rac1 dependency

• Cytokine assessment:

  • Collect supernatants at various time points post-stimulation

  • Use multiplex cytokine arrays or individual ELISAs to measure TNF-α, MCP-1, and other inflammatory mediators

• Signaling pathway analysis:

  • Prepare cell lysates at early time points post-stimulation

  • Use ELMO1 Antibody, HRP conjugated to confirm ELMO1 expression

  • Probe for phosphorylated NFκB, ERK1/2, and p38 MAPK

Research has demonstrated that ELMO1-depleted macrophages show reduced release of proinflammatory cytokines, including TNF-α and MCP-1. This reduction correlates with impaired activation of key signaling pathways, including NFκB, ERK1/2, and p38 MAPK .

What controls should be included when using ELMO1 Antibody, HRP conjugated in research studies?

When using ELMO1 Antibody, HRP conjugated, researchers should include several controls to ensure result validity:

• Positive control:

  • Known ELMO1-expressing samples like human placenta tissue lysate

  • Cell lines with confirmed ELMO1 expression such as Jurkat cells or H1299 cells

• Negative controls:

  • ELMO1 knockout/knockdown samples (if available)

  • Isotype controls to assess non-specific binding

  • Peptide competition assays using the immunizing peptide

• Loading controls:

  • Housekeeping proteins (β-actin, GAPDH, etc.) to ensure equal loading

  • Total protein staining methods (Ponceau S, SYPRO Ruby) as alternatives

• Molecular weight verification:

  • The predicted molecular weight of human ELMO1 is 84 kDa

  • Include molecular weight markers to confirm detection of correctly sized protein

• Experimental validation controls:

  • When studying ELMO1-dependent processes, include both genetic (knockdown/knockout) and pharmacological (Rac1 inhibitors) approaches

  • For functional studies, rescue experiments by re-expressing ELMO1 in depleted cells

These controls will help ensure specificity, reproducibility, and biological relevance of findings.

How can ELMO1 Antibody, HRP conjugated be used to investigate ELMO1's role in renal protection?

ELMO1 has been identified as an important factor for glomerular protection and renal cell survival, particularly under diabetic conditions. To investigate this role:

• Model systems:

  • In vitro: Renal cell lines (podocytes, tubular epithelial cells) under normal and high glucose conditions

  • In vivo: Diabetic mouse models, zebrafish models (where ELMO1 overexpression rescued hyperglycemia-induced renal pathology)

• Apoptosis assessment:

  • Compare apoptosis rates in control versus ELMO1-depleted renal cells

  • Use TUNEL assays, Annexin V staining, or caspase activation assays

  • Correlate with ELMO1 expression levels using Western blot with ELMO1 Antibody, HRP conjugated

• Rescue experiments:

  • Overexpress ELMO1 in diabetic models to assess protective effects

  • Use apoptosis inhibitors to determine if they rescue the phenotype of ELMO1 deficiency

• Human sample analysis:

  • Evaluate ELMO1 expression in kidney biopsies from non-diabetic and diabetic patients

  • Use immunohistochemistry to localize ELMO1 in glomerular podocytes and tubules

  • Correlate expression patterns with disease severity

Research has shown that ELMO1 protects renal structure and ultrafiltration in kidney development and disease by decreasing apoptosis. In zebrafish models, ELMO1 overexpression rescued hyperglycemia-induced pathophysiological alterations in the pronephros .

What are common technical issues when using ELMO1 Antibody, HRP conjugated and how can they be resolved?

When working with ELMO1 Antibody, HRP conjugated, researchers may encounter several technical challenges:

• Weak or no signal:

  • Increase antibody concentration within recommended range (1:300-5000)

  • Extend incubation time (overnight at 4°C)

  • Increase protein loading (up to 30 μg of total protein)

  • Use enhanced chemiluminescence substrate for higher sensitivity

  • Check storage conditions and antibody expiration date

• High background:

  • Increase blocking intensity (use 5% BSA instead of 2%)

  • Optimize antibody dilution (try more diluted concentrations)

  • Reduce incubation time or temperature

  • Ensure thorough washing between steps (use 5-6 washes with TBST)

• Multiple bands:

  • Verify ELMO1 isoforms in your sample (potential splice variants)

  • Check for protein degradation by adding protease inhibitors

  • Assess antibody specificity using knockout/knockdown controls

• Inconsistent results:

  • Standardize protein extraction methods

  • Use freshly prepared samples when possible

  • Aliquot antibody to avoid repeated freeze-thaw cycles

  • Maintain consistent transfer conditions for Western blot

ELMO1 Antibody, HRP conjugated should detect a band of approximately 84 kDa in Western blots . If experiencing persistent issues, validating with a non-conjugated ELMO1 antibody and standard secondary antibody approach may help troubleshoot whether the problem lies with the primary antibody specificity or the HRP conjugation.

How can researchers optimize ELMO1 Antibody, HRP conjugated for detecting low expression levels?

For detecting low levels of ELMO1 expression, researchers can employ several optimization strategies:

• Sample preparation:

  • Enrich for ELMO1 through immunoprecipitation before Western blot

  • Use phosphatase inhibitors along with protease inhibitors in lysis buffers

  • Consider subcellular fractionation to concentrate cytoplasmic proteins

• Blotting technique:

  • Use PVDF membrane instead of nitrocellulose for higher protein binding capacity

  • Reduce transfer voltage and extend transfer time (e.g., 30V for 70 minutes)

  • Use a semi-dry transfer system for efficient transfer of mid-sized proteins

• Detection enhancement:

  • Employ high-sensitivity ECL substrate

  • Extend exposure time gradually to capture weak signals

  • Use cooled CCD camera systems for higher sensitivity and dynamic range

  • Consider tyramide signal amplification techniques

• Antibody optimization:

  • Test concentration at the lower end of the dilution range (1:300-1:1000)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use a signal enhancer solution before antibody incubation

These optimizations can significantly improve detection sensitivity for samples with low ELMO1 expression, such as primary cells or tissue samples with limited material availability.

How can ELMO1 Antibody, HRP conjugated be used in investigating neutrophil function in arthritis models?

Recent research has revealed a non-canonical role for ELMO1 as a cytoplasmic regulator of specific neutrophil receptors and as a promoter of arthritis . To investigate this relationship:

• Neutrophil isolation:

  • Isolate neutrophils from wild-type and ELMO1-deficient mice (using Elmo1 fl/fl Mrp8-Cre mice for neutrophil-specific deletion)

  • Alternative approach: isolate neutrophils from peripheral blood of human donors with ELMO1 SNP associated with arthritis

• Migration and function assays:

  • Assess neutrophil migration to arthritis-related chemokines

  • Evaluate neutrophil infiltration into joint tissues using histological approaches

  • Measure reactive oxygen species production and NET formation

• Expression analysis:

  • Use ELMO1 Antibody, HRP conjugated to confirm ELMO1 expression levels in neutrophils

  • Compare expression between control and arthritic conditions

• In vivo models:

  • K/BxN serum-induced arthritis model in control and ELMO1-deficient mice

  • Measure joint inflammation, clinical scores, and histopathology

  • Correlate neutrophil recruitment with disease severity

Studies have shown that ELMO1-deficient mice exhibit reduced joint inflammation in acute and chronic arthritis models, and neutrophils from human donors carrying the SNP in ELMO1 associated with arthritis display increased migratory capacity .

What are promising future research directions for ELMO1 using this antibody?

Several promising research directions for ELMO1 investigation using ELMO1 Antibody, HRP conjugated include:

• Therapeutic targeting:

  • Screening of small molecule inhibitors targeting ELMO1-DOCK1 interaction

  • Evaluation of ELMO1 inhibition as a therapeutic strategy for inflammatory conditions

  • Development of peptide-based inhibitors of ELMO1 function

• Disease associations:

  • Further investigation of ELMO1's role in diabetic nephropathy

  • Exploring ELMO1 function in other autoimmune conditions beyond arthritis

  • Assessment of ELMO1 polymorphisms in disease susceptibility

• Mechanistic studies:

  • Detailed mapping of ELMO1-dependent signaling networks using phosphoproteomics

  • Investigation of post-translational modifications of ELMO1 that regulate its function

  • Characterization of additional ELMO1 binding partners in cell type-specific contexts

• Structural biology applications:

  • Combining antibody-based detection with structural studies to identify functional domains

  • Using epitope mapping to understand structure-function relationships

• Single-cell applications:

  • Adapting ELMO1 antibodies for single-cell analysis techniques

  • Investigating cell-to-cell variability in ELMO1 expression and function

These research directions represent opportunities to expand our understanding of ELMO1 biology and its potential as a therapeutic target in various disease contexts.