ELMO3 Antibody

What is ELMO3 and why is it important in cancer research?

ELMO3 is a member of the engulfment and cell motility (ELMO) protein family that plays a vital role in the processes of chemotaxis and metastasis of tumor cells. The importance of ELMO3 stems from its significantly higher expression in various cancer tissues compared to adjacent normal tissues, as demonstrated in multiple studies of gastric cancer patients . The protein has been shown to participate in crucial cellular processes including cell proliferation, invasion, migration, cell cycle regulation, and F-actin polymerization, making it a potential diagnostic and prognostic marker for various cancers . Research has shown that ELMO3 expression is particularly elevated in patients with lymph node metastasis, suggesting its involvement in cancer progression and spread . Additionally, ELMO3 has been identified as having a positive correlation with COX-2 expression in non-small cell lung cancer (NSCLC) tissues, indicating potential involvement in inflammatory pathways that promote cancer development .

What types of ELMO3 antibodies are available for research, and how do they differ?

The most common type of ELMO3 antibody available for research is the polyclonal rabbit IgG antibody, which has been validated for human samples and predicted to react with mouse and rat samples with high sequence homology (91% and 93% respectively) . These antibodies are typically produced against recombinant protein corresponding to specific amino acid sequences of ELMO3, such as "LVKSEVPLDRLLVHLQVMNQQLQTKAMALLTALLQGASPVERKHMLDYLWQRNLRQFIYKNIIHSAAPMGDEMAHHLYVLQALMLGL" . Commercial ELMO3 antibodies are available in unconjugated formats, with concentrations that may vary between lots . The differences between available antibodies primarily relate to their specificity, sensitivity, and validated applications rather than structural differences. Current commercially available ELMO3 antibodies have been validated for techniques including immunocytochemistry/immunofluorescence, immunohistochemistry, and immunohistochemistry-paraffin applications . The selection of a specific ELMO3 antibody should be based on the intended application, species of interest, and the specific epitope being targeted within the ELMO3 protein structure.

What are the validated applications for ELMO3 antibodies in cancer research?

ELMO3 antibodies have been validated for several critical applications in cancer research, primarily focusing on protein detection and localization in various cellular contexts. Immunocytochemistry and immunofluorescence applications have successfully demonstrated the localization of ELMO3 to plasma membrane and cell junctions in human cell lines such as A-431, providing insights into its functional role in cellular adhesion and migration . Immunohistochemistry applications, particularly in paraffin-embedded tissues, have shown strong ELMO3 positivity in specific cell types, such as Leydig cells in human testis, indicating tissue-specific expression patterns that may be relevant to cancer development . In gastric cancer research, ELMO3 antibodies have been employed in western blot analysis to compare expression levels between tumor tissues and adjacent normal tissues, revealing significantly higher expression in cancerous samples . The antibodies have also been used to investigate the correlation between ELMO3 expression and clinical pathological features, such as lymph node metastasis status, demonstrating their utility in translational research connecting molecular findings to clinical outcomes . Additionally, researchers have utilized ELMO3 antibodies to evaluate the efficiency of siRNA-mediated knockdown in functional studies investigating cell proliferation, migration, and invasion in cancer cell lines .

How should ELMO3 antibody be used for immunohistochemistry of paraffin-embedded tissues?

For optimal immunohistochemistry of paraffin-embedded tissues using ELMO3 antibody, researchers should follow a rigorous protocol that begins with proper sample preparation. Tissue specimens should be fixed in 10% neutral buffered formalin for 24-48 hours, processed, and embedded in paraffin according to standard protocols. Sections should be cut at 4-6 μm thickness and mounted on positively charged slides to ensure adhesion throughout the staining procedure . Antigen retrieval is a critical step that typically involves heating the deparaffinized sections in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 15-20 minutes, as this helps to unmask epitopes that may have been cross-linked during fixation and improves antibody binding. When working with ELMO3 antibody specifically, blocking endogenous peroxidase activity with 3% hydrogen peroxide and preventing non-specific binding with serum-free protein block are essential steps before applying the primary antibody . The primary ELMO3 antibody should be diluted according to manufacturer recommendations (typically 1:100 to 1:500) and incubated overnight at 4°C or for 1-2 hours at room temperature in a humidified chamber. After thorough washing with PBS or TBS buffer, an appropriate secondary antibody system should be applied, followed by detection with DAB or other chromogen systems . Counterstaining with hematoxylin provides contrast to visualize cellular morphology while evaluating ELMO3 expression patterns. Successful ELMO3 staining in paraffin sections can reveal its expression in specific cellular compartments, such as the strong positivity observed in Leydig cells of the testis, which serves as a positive control for the technique .

What are the best experimental conditions for using ELMO3 antibody in immunofluorescence assays?

Optimizing experimental conditions for ELMO3 antibody in immunofluorescence assays requires attention to several critical factors. Cells should be cultured on glass coverslips or chamber slides coated with appropriate substrate (e.g., poly-L-lysine or collagen) to promote adhesion while maintaining normal morphology. Fixation method significantly impacts epitope preservation and accessibility; for ELMO3 detection, 4% paraformaldehyde for 15 minutes at room temperature typically yields good results while preserving membrane structures where ELMO3 is often localized . Permeabilization should be gentle (0.1-0.2% Triton X-100 for 5-10 minutes) to allow antibody access to intracellular epitopes without disrupting membrane structures. Non-specific binding sites must be blocked with 1-5% BSA or 5-10% normal serum from the species in which the secondary antibody was raised, typically for 30-60 minutes at room temperature . The primary ELMO3 antibody should be diluted according to validated recommendations (usually 1:100 to 1:500) in blocking buffer and incubated overnight at 4°C or for 2-3 hours at room temperature in a humidified chamber. After thorough washing with PBS (at least 3 times for 5 minutes each), fluorophore-conjugated secondary antibodies should be applied at appropriate dilutions (typically 1:200 to 1:1000) and incubated for 1 hour at room temperature in the dark . Counter-staining with DAPI (1:1000) for 5 minutes allows visualization of nuclei, while phalloidin staining can provide information about F-actin structures that may interact with ELMO3. Mounting should be done with anti-fade mounting medium to prevent photobleaching during microscopy. This protocol has successfully demonstrated ELMO3 localization to plasma membrane and cell junctions in human cell lines, providing valuable spatial information about its potential functional roles .

How can ELMO3 antibody be used to investigate cancer cell migration and invasion mechanisms?

ELMO3 antibody can be strategically employed to investigate cancer cell migration and invasion through multiple complementary approaches. Researchers can utilize ELMO3 antibody for immunofluorescence staining to visualize its co-localization with cytoskeletal elements involved in cell motility, particularly at the leading edge of migrating cells where dynamic F-actin polymerization occurs . This technique has revealed that ELMO3 often localizes to plasma membrane and cell junctions, suggesting its involvement in cell-cell adhesion dynamics during collective migration . In mechanistic studies, ELMO3 antibody can be used in combination with siRNA-mediated knockdown experiments to validate the specificity of phenotypic changes observed in wound-healing and Transwell migration/invasion assays . The antibody can confirm the reduction in ELMO3 protein levels following siRNA treatment, which has been shown to significantly suppress cell mobility, migration, and invasion in gastric cancer cell lines like SGC7901 and BGC823 . For more advanced applications, ELMO3 antibody can be employed in immunoprecipitation experiments to identify novel protein-protein interactions that may regulate the migratory and invasive capabilities of cancer cells, similar to how COX-2 has been shown to interact with the related protein ELMO1 . Western blot analysis using ELMO3 antibody can quantify changes in protein expression following treatment with potential therapeutic agents, such as COX-2 inhibitors, which have been demonstrated to down-regulate ELMO3 expression in NSCLC models . Combined with functional assays measuring invasion through Matrigel basement membrane, these antibody-based techniques provide comprehensive tools for dissecting the molecular mechanisms by which ELMO3 promotes cancer metastasis.

What techniques can be used to study the interaction between ELMO3 and other signaling proteins in cancer pathways?

Studying the interactions between ELMO3 and other signaling proteins in cancer pathways requires sophisticated techniques that preserve native protein complexes while providing specific detection. Co-immunoprecipitation (co-IP) using ELMO3 antibody represents a fundamental approach, allowing researchers to pull down ELMO3 along with its binding partners for subsequent analysis by western blotting or mass spectrometry . This technique has been valuable in studying similar interactions, such as the documented association between COX-2 and ELMO1, suggesting potential parallel mechanisms for ELMO3 given their structural similarities, including shared domains like the Ras GTPase-binding domain (RBD), ELMO Inhibitor domain (EID), and ELMO autoregulatory domain (EAD) . Proximity ligation assay (PLA) offers a more sensitive approach for detecting protein interactions in situ, enabling visualization of ELMO3 interactions with candidate proteins within the cellular context, particularly valuable for examining connections to RhoG and Rac1 signaling components that have been implicated in ELMO family function . For tracking dynamic interactions in living cells, bimolecular fluorescence complementation (BiFC) or fluorescence resonance energy transfer (FRET) approaches using tagged ELMO3 in combination with antibody validation can reveal interaction kinetics and subcellular localization during processes like cell migration. Pull-down assays using recombinant ELMO3 domains, particularly the RBD that has been crystallized in related proteins, can identify specific binding regions involved in protein-protein interactions . These approaches collectively provide complementary data on how ELMO3 interacts with cytoskeletal regulators, adhesion molecules, and signaling components to promote cancer cell invasion and metastasis, potentially revealing novel therapeutic targets within these interaction networks.

How can ELMO3 antibody be used in developing potential therapeutic strategies for cancer?

ELMO3 antibody can be instrumental in developing potential therapeutic strategies for cancer through multiple research avenues. In target validation studies, the antibody enables precise quantification of ELMO3 protein levels in patient-derived samples, facilitating stratification of patients who might benefit from ELMO3-targeted therapies based on expression levels that correlate with metastatic potential . Researchers can employ ELMO3 antibody in high-throughput screening assays to identify small molecules or biologics that disrupt ELMO3's interactions with binding partners or alter its subcellular localization, similar to approaches targeting other members of the ELMO family like ELMO1's RBD domain . The antibody is essential for confirming the specificity and efficacy of potential therapeutic agents such as siRNAs or antisense oligonucleotides designed to reduce ELMO3 expression, which has been shown to inhibit processes of cell proliferation, invasion, metastasis, and cell cycle progression in gastric cancer and NSCLC models . For developing antibody-drug conjugates (ADCs), anti-ELMO3 antibodies could potentially be conjugated with cytotoxic payloads to selectively target cancer cells with high ELMO3 expression, though this application would require antibodies with high specificity for cell-surface exposed epitopes. In monitoring therapeutic response, ELMO3 antibody enables assessment of target engagement and downstream pathway modulation through immunohistochemistry of tumor biopsies or circulating tumor cells before and after treatment . Additionally, researchers investigating indirect approaches, such as COX-2 inhibition with parecoxib (which has been shown to down-regulate ELMO3 expression), can use the antibody to monitor changes in ELMO3 levels and correlate them with inhibition of EMT, adhesion, and metastasis in experimental models .

What are common challenges in ELMO3 antibody experiments and how can they be addressed?

Researchers working with ELMO3 antibodies may encounter several technical challenges that require specific troubleshooting approaches. One common issue is weak or absent signal in immunostaining applications, which can result from inadequate antigen retrieval in fixed tissues. This can be addressed by optimizing retrieval methods, such as extending heating time in citrate buffer or testing alternative pH buffers (EDTA pH 9.0 instead of citrate pH 6.0), as different fixation protocols may affect epitope accessibility . Non-specific background staining presents another challenge, particularly in immunohistochemistry, which can be mitigated by increasing blocking time with serum-free protein block, using more dilute antibody concentrations, or adding 0.1-0.3% Triton X-100 to reduce non-specific hydrophobic interactions . Inconsistent results between experiments often stem from variability in antibody quality between lots; researchers should validate new lots against previous successful experiments and consider using pooled positive control samples as internal standards. When working with cell lines, researchers may face difficulties detecting endogenous ELMO3 due to variable expression levels; this can be addressed by first screening multiple cell lines (such as SGC7901, BGC823, MGC803, AGS, and MKN74 for gastric cancer studies) to identify those with higher baseline expression . Cross-reactivity with other ELMO family members (particularly ELMO1 and ELMO2) can complicate data interpretation due to structural similarities; researchers should validate antibody specificity using knockdown controls, recombinant protein competition assays, or by probing tissues known to differentially express ELMO family members . For western blot applications, degradation of ELMO3 during sample preparation can lead to multiple bands or weak signals; this can be prevented by working rapidly on ice, using fresh protease inhibitor cocktails, and optimizing lysis buffer compositions specific to membrane-associated proteins.

How can researchers differentiate between specific and non-specific staining when using ELMO3 antibody?

Differentiating between specific and non-specific staining when using ELMO3 antibody requires implementation of rigorous controls and validation strategies. Primary negative controls, where the primary antibody is omitted but all other steps remain identical, help identify background staining from the detection system or endogenous enzymes/biotin. Isotype controls using non-specific IgG from the same species and at the same concentration as the ELMO3 antibody can reveal background caused by non-specific antibody binding or Fc receptor interactions . Peptide competition or blocking experiments, where the ELMO3 antibody is pre-incubated with excess immunizing peptide (such as the recombinant protein fragment used to generate the antibody) before application to samples, should eliminate specific staining while leaving non-specific signals unchanged . Positive tissue controls with known ELMO3 expression patterns, such as human testis where strong positivity has been documented in Leydig cells, provide crucial reference points for confirming staining specificity . Genetic validation approaches using siRNA knockdown samples processed in parallel with untreated samples provide powerful evidence of antibody specificity, as the staining intensity should correlate with protein reduction levels verified by western blot . Cross-validation between different detection methods (e.g., comparing immunofluorescence localization with immunohistochemistry or western blot results) strengthens confidence in staining patterns, particularly when examining membranous and junctional localization patterns reported for ELMO3 . Finally, researchers should compare staining patterns with published literature on ELMO3 localization and evaluate whether the observed subcellular distribution aligns with its known functional roles in cell migration, invasion, and cytoskeletal organization processes .

How can ELMO3 antibody be used to investigate the relationship between COX-2 inhibition and cancer metastasis?

ELMO3 antibody provides a powerful tool for investigating the recently discovered relationship between COX-2 inhibition and cancer metastasis. Researchers can utilize ELMO3 antibody in western blot or immunohistochemistry analyses to quantify changes in ELMO3 protein expression following treatment with COX-2 inhibitors like parecoxib in both in vitro cancer cell models and in vivo tumor xenografts . This approach has already revealed that COX-2 inhibitors can significantly down-regulate ELMO3 expression, suggesting a molecular link between these two pathways in cancer progression . Dual immunofluorescence staining with antibodies against both ELMO3 and COX-2 can elucidate their spatial relationship and potential co-localization in tumor tissues, complementing correlation analyses that have demonstrated a positive relationship between these proteins' expression levels in NSCLC tissues . To investigate the molecular mechanism connecting these pathways, researchers can employ ELMO3 antibody in co-immunoprecipitation experiments to determine whether COX-2 directly interacts with ELMO3, similar to the documented interaction between COX-2 and the related protein ELMO1 . Time-course experiments monitoring ELMO3 expression changes following COX-2 inhibition can reveal the kinetics of this regulatory relationship, potentially identifying the optimal therapeutic window for intervention. Functional assays measuring cancer cell adhesion, invasion, and metastasis following COX-2 inhibitor treatment should be accompanied by ELMO3 antibody detection to establish whether the degree of ELMO3 downregulation correlates with inhibition of these metastatic processes . Additionally, rescue experiments where ELMO3 is overexpressed in COX-2 inhibitor-treated cells can determine whether persistent ELMO3 expression can overcome the anti-metastatic effects of COX-2 inhibition, with ELMO3 antibody detection confirming successful restoration of protein levels .

What are the prospects for developing new ELMO3-targeting therapeutic antibodies for cancer treatment?

The development of ELMO3-targeting therapeutic antibodies represents a promising frontier in cancer treatment, building on emerging understanding of ELMO3's role in tumor progression. Unlike conventional research-grade antibodies that primarily serve detection purposes, therapeutic antibodies would need to specifically bind ELMO3 epitopes in a way that disrupts its function in promoting cell migration, invasion, and proliferation . A critical first step in this development would be structural characterization of accessible ELMO3 domains, similar to how the Ras-binding domain (RBD) structure has been determined for the related ELMO1 protein, which served as a basis for successful nanobody design . While ELMO3 is primarily an intracellular protein involved in cytoskeletal regulation, certain domains may be transiently exposed during cell migration or may be accessible through novel delivery approaches such as antibody-drug conjugates targeting cancer cells with high ELMO3 expression . Computer-aided design approaches similar to the dock-and-design methodology used for ELMO1-RBD could accelerate the development of high-affinity antibody fragments specifically targeting ELMO3 functional domains . The therapeutic potential is supported by consistent findings that ELMO3 silencing through siRNA significantly inhibits cancer cell growth, invasion, and metastasis in multiple cancer types, suggesting that antibody-mediated neutralization might achieve similar clinical benefits . Because ELMO3 expression is significantly elevated in cancer tissues compared to normal tissues, therapeutic antibodies could potentially achieve a favorable therapeutic window with minimal off-target effects . Future development efforts should focus on identifying the most functionally critical and accessible epitopes within ELMO3, optimizing antibody format (conventional antibodies, single-domain antibodies, or bispecific constructs), and evaluating combination approaches with existing therapies such as COX-2 inhibitors that have shown synergistic potential through ELMO3 downregulation .

How might ELMO3 antibody be used in the development of companion diagnostics for cancer treatment selection?

ELMO3 antibody holds significant potential in the development of companion diagnostics for cancer treatment selection, particularly as therapies targeting cell motility and invasion pathways advance to clinical testing. Immunohistochemistry using validated ELMO3 antibodies could be standardized to quantitatively assess ELMO3 expression levels in tumor biopsies, providing a predictive biomarker to identify patients most likely to benefit from therapies targeting ELMO3 directly or indirectly through related pathways . The established correlation between ELMO3 overexpression and lymph node metastasis in gastric cancer patients suggests that ELMO3 immunohistochemistry scoring could stratify patients according to metastatic risk, potentially guiding decisions about adjuvant therapy intensity . Development of an ELMO3 antibody-based companion diagnostic would require rigorous validation across multiple cancer types, establishing clinically relevant cutoff values that correlate with treatment response, similar to established companions for targeted therapies like HER2 testing for trastuzumab treatment . For monitoring treatment response, ELMO3 antibody-based liquid biopsy assays could potentially detect ELMO3-expressing circulating tumor cells, providing a minimally invasive method to assess ongoing treatment efficacy. The positive correlation between ELMO3 and COX-2 expression in NSCLC suggests that ELMO3 immunohistochemistry might identify patients most likely to benefit from COX-2 inhibitor therapy, with potential applications in selection for clinical trials investigating parecoxib or similar agents in cancer treatment . Multiplex immunohistochemistry platforms combining ELMO3 antibody with antibodies against other markers (such as epithelial-mesenchymal transition markers like E-cadherin, N-cadherin, and Vimentin) could provide a more comprehensive assessment of a tumor's invasive and metastatic potential, enhancing treatment selection precision . As with all companion diagnostics, development would require prospective clinical trials demonstrating that ELMO3 antibody-based testing can effectively identify patient populations with differential benefit from specific therapeutic approaches, ultimately improving outcomes through personalized treatment selection.