EML4 Antibody

What is EML4 and what cellular functions does it perform?

EML4 (echinoderm microtubule associated protein like 4) is a 120 kDa protein essential for microtubule formation and stability. It plays critical roles in cellular organization, particularly during cell division. EML4 is required for proper organization of the mitotic spindle and for the correct attachment of kinetochores to microtubules . Additionally, this protein promotes the recruitment of NUDC to the mitotic spindle, which is necessary for mitotic progression . Within signaling pathways, EML4 interacts with key proteins like KRAS and BRAF, contributing to signal transduction that controls cell proliferation and differentiation . The protein is also known by several synonyms including C2orf2, ELP120, EMAP-4, EMAPL4, and ROPP120 .

What applications are supported for EML4 antibodies and which cell lines show positive reactivity?

EML4 antibodies can be used in multiple experimental applications as evidenced by validation data. The primary applications include:

Positive reactivity has been demonstrated in several human cell lines, including:

• A549 cells (for WB, IP, and IF/ICC)

• A431 cells (for WB)

• HeLa cells (for WB)

Additionally, EML4 antibodies have shown positive immunohistochemical detection in human tissue samples including breast cancer tissue, prostate cancer tissue, and lung cancer tissue .

How should EML4 antibodies be stored and handled to maintain optimal performance?

For optimal antibody performance, EML4 antibodies should be stored at -20°C where they remain stable for one year after shipment . Most commercial antibodies are supplied in a storage buffer consisting of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For antibodies supplied in this format, aliquoting is generally unnecessary for -20°C storage, which simplifies laboratory handling procedures .

Smaller size preparations (e.g., 20μl) may contain 0.1% BSA as a stabilizer . When working with the antibody, avoid repeated freeze-thaw cycles as this can degrade antibody performance. For immunohistochemical applications, antigen retrieval using TE buffer at pH 9.0 is typically recommended, although citrate buffer at pH 6.0 may alternatively be used for certain applications .

What is the EML4-ALK fusion gene and why is it significant in cancer research?

The EML4-ALK fusion gene results from a chromosomal rearrangement (inversion within chromosome 2p) that creates a fusion between the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) genes . This genetic alteration is particularly significant in non-small cell lung carcinoma (NSCLC) research and treatment strategies .

Multiple variants of the EML4-ALK fusion have been identified, with variants 1 and 3 being the most commonly studied . The fusion gene produces an oncogenic protein that drives cancer cell proliferation and survival. The significance of this fusion lies in:

• Its role as a driver mutation in a subset of NSCLC cases

• Its status as a therapeutic target for ALK inhibitors

• Its utility as a biomarker for patient selection for targeted therapies

How can researchers validate the specificity of EML4 antibodies?

Validation of EML4 antibodies requires a multi-faceted approach to ensure specificity and reliability:

• CRISPR-Cas9 edited cell lines: One robust validation method involves testing the antibody in wild-type cells (e.g., HeLa) versus EML4 CRISPR-Cas9 edited cell lines. Loss of signal in the edited cell line confirms specificity, as demonstrated with antibodies like ab85834 .

• Positive control systems: Using cells transfected with EML4 expression constructs serves as an effective positive control. For example, Phoenix cells transfected with pcDNA3_EML4-ALK can be used as positive controls for expression of EML4-ALK variant 1 .

• Western blot analysis: Verification of the antibody's ability to detect the expected molecular weight band (approximately 120 kDa for EML4) across multiple cell lines provides evidence of specificity.

• Immunoprecipitation followed by mass spectrometry: This approach can verify that the antibody captures the intended target protein and identifies any potential cross-reactivity.

• Cell line panels: Testing across multiple cell lines with known EML4 expression profiles (such as A549, A431, and HeLa cells) provides further validation of specificity and application range.

What methodological considerations are crucial when using EML4 antibodies for detecting EML4-ALK fusion proteins?

When using EML4 antibodies to detect EML4-ALK fusion proteins, researchers must consider several critical methodological factors:

• Variant specificity: The EML4-ALK fusion gene exists in multiple variants (such as variants 1 and 3) , which produce fusion proteins of different sizes. Antibodies may have differential reactivity to these variants, so understanding which variant(s) your antibody detects is essential for proper experimental design and data interpretation.

• Expression heterogeneity: Studies have shown that only a minority of cells may harbor the EML4-ALK gene in some specimens . This heterogeneity necessitates careful sampling and analysis approaches, including examination of multiple tumor regions in clinical specimens.

• Signal interpretation thresholds: For immunohistochemical detection, establishing appropriate positive signal thresholds is crucial. Research indicates that cells can be considered positive for ALK if >5% of cells show cytoplasmic staining of at least grade 1 intensity (on a scale of 0 to 3) . Clear documentation of scoring criteria is essential for reproducible results.

• Verification with complementary techniques: Due to potential discrepancies between transcript detection and protein expression , verification using multiple detection methods (e.g., FISH, RT-PCR, and immunohistochemistry) is recommended for conclusive identification of EML4-ALK positive cases.

• Subcellular localization consideration: EML4-ALK forms cytoplasmic protein condensates , so immunofluorescence protocols should be optimized to detect these structures, which may have different staining patterns than diffusely expressed proteins.

How do detection methods for EML4-ALK compare in terms of sensitivity, specificity, and cost-effectiveness?

Comparison of detection methods for EML4-ALK reveals important differences in performance metrics:

Immunohistochemistry (IHC) using antibodies directed against the EML4-ALK fusion protein provides a widely available alternative method of detection . The performance of IHC depends significantly on the antibody clone used. For instance, the 5A4 monoclonal antibody has demonstrated good correlation with FISH results in multiple studies .

Cost analysis studies have suggested that using ALK IHC as a screening test followed by confirmatory FISH testing only for IHC-positive or equivocal cases can be a cost-effective approach in clinical settings . This tiered testing approach balances sensitivity, specificity, and resource utilization.

Notably, research has shown discrepancies between transcript detection and protein expression. In some studies, EML4-ALK transcripts were detected by RT-PCR in samples where no protein expression was detectable by IHC, Western blotting, or immunoprecipitation , highlighting the need for careful method selection based on the specific research question.

What challenges exist in interpreting EML4-ALK expression in clinical samples and how can researchers address them?

Interpreting EML4-ALK expression in clinical samples presents several challenges that researchers must navigate:

• Non-specific expression: EML4-ALK transcripts (variants 1 and 3) have been detected in non-cancerous lung tissues taken far from tumors, suggesting transcript detection alone may not be tumor-specific . This raises concerns about using transcript detection as a singular diagnostic approach.

• Expression heterogeneity: Studies have demonstrated that only a minority of cells may harbor the EML4-ALK gene in some specimens . This heterogeneity can lead to sampling errors and false negatives if examined tissue sections do not contain the positive cell populations.

• Discrepancies between transcript and protein detection: Some samples positive for EML4-ALK transcripts have shown no detectable protein expression when examined by immunohistochemistry, Western blotting, and immunoprecipitation . This disconnect complicates the interpretation of molecular testing results.

• Technical artifacts: For IHC, interpretation challenges include distinguishing true positive staining from background or non-specific staining, particularly in samples with mucin production or pigment deposition.

To address these challenges, researchers should:

• Implement multi-modality testing combining FISH, IHC, and molecular methods

• Establish clear scoring criteria for IHC (e.g., >5% of cells showing cytoplasmic staining of at least grade 1 intensity)

• Use appropriate positive and negative controls with each batch of testing

• Consider tissue microenvironment and fixation variables when interpreting results

• Document heterogeneity patterns when observed to inform clinical decision-making

How does EML4 contribute to mitotic spindle organization and what techniques can be used to study this function?

EML4 plays essential roles in mitotic spindle organization through several mechanisms:

• Microtubule formation and stability: EML4 is essential for the formation and stability of microtubules , which are the primary structural components of the mitotic spindle.

• Kinetochore-microtubule attachment: Research has demonstrated that EML4 is required for the proper attachment of kinetochores to microtubules during mitosis . This attachment is crucial for correct chromosome segregation.

• NUDC recruitment: EML4 promotes the recruitment of NUDC (Nuclear Distribution protein C) to the mitotic spindle, which is necessary for mitotic progression .

To study these functions, researchers can employ several advanced techniques:

• Live-cell imaging with fluorescently tagged EML4: This approach allows visualization of EML4 dynamics during mitosis and can be combined with other markers to study co-localization patterns.

• Optogenetic manipulation: As used in recent studies of EML4-ALK assemblies , optogenetic approaches enable temporal control of protein activation or inhibition to study dynamic processes during mitosis.

• CRISPR-Cas9 genome editing: Creating EML4 knockout or domain-specific mutant cell lines helps determine the specific contributions of EML4 domains to spindle organization.

• Proximity labeling techniques (BioID or APEX): These can identify proteins in close proximity to EML4 during mitosis, revealing the protein interaction network involved in spindle assembly.

• Super-resolution microscopy: Techniques such as SIM, STED, or STORM provide nanoscale resolution of EML4 localization relative to microtubules and kinetochores during different mitotic stages.

What are the latest findings on EML4-ALK protein assemblies and their implications for therapeutic resistance?

Recent research has unveiled important insights into EML4-ALK protein assemblies and their relationship to therapeutic resistance:

EML4-ALK has been found to form cytoplasmic protein condensates, which result from networks of interactions between oncogene and adapter protein multimers . These assemblies are associated with oncogenic signaling, though their precise role in drug response has been an area of active investigation.

New studies using optogenetics and live-cell imaging have revealed that EML4-ALK assemblies suppress transmembrane receptor tyrosine kinase (RTK) signaling . This finding has significant implications for understanding resistance mechanisms to ALK inhibitors, as RTK activation is a known bypass mechanism that can allow cancer cells to survive despite ALK inhibition.

The formation of these cytoplasmic protein condensates represents a structural organization that may influence drug accessibility to the kinase domain of ALK. This spatial organization could contribute to differential responses to ALK inhibitors among patients and the eventual development of resistance.

These assemblies may also create microenvironments with concentrated signaling components that amplify oncogenic signaling pathways, potentially explaining the potent transforming ability of EML4-ALK fusion proteins even when expressed at relatively low levels.

Understanding these assembly dynamics opens new avenues for therapeutic approaches that might target not just the ALK kinase activity but also the formation or stability of these protein assemblies, potentially overcoming resistance mechanisms that emerge during treatment with conventional ALK inhibitors.

What are the optimal sample preparation protocols for different EML4 antibody applications?

For optimal results with EML4 antibodies across different applications, researchers should follow these sample preparation guidelines:

Western Blotting:

• Cell lysis should be performed using a buffer containing 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1% Triton X-100, 0.5% deoxycholic acid (DOC), 0.1% SDS, and 1 mmol/L sodium orthovanadate, plus a protease inhibitor cocktail (leupeptin, aprotinin, pepstatin A, and pefabloc) .

• For optimal separation of the 120 kDa EML4 protein, use 7.5-10% polyacrylamide gels.

• Transfer proteins to polyvinylidene difluoride membranes for best results with EML4 detection .

• Block membranes with 3-5% milk or BSA in TBS-T (0.1% Tween) .

Immunohistochemistry:

• For formalin-fixed, paraffin-embedded tissues, perform antigen retrieval using either:

  • TE buffer at pH 9.0 (primary recommendation) or

  • Citrate buffer at pH 6.0 (alternative option)

• Microwave heating (750-W, three 5-minute cycles) is recommended for effective retrieval .

• For detection systems, either alkaline-phosphatase/RED (Dako-REAL) or DAB-based (Envision + DAB) systems have been validated .

Immunofluorescence:

• Fix cells using either 4% paraformaldehyde (for structural preservation) or methanol (for better access to microtubule-associated proteins).

• Permeabilize with 0.1-0.5% Triton X-100 for optimal antibody access to intracellular EML4.

• Block with 1-5% BSA or normal serum from the same species as the secondary antibody.

Immunoprecipitation:

• Use 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate .

• Pre-clear lysates with appropriate control IgG and protein A/G beads to reduce non-specific binding.

• Include both positive controls (known EML4-expressing cells like A549) and negative controls in experimental design .

How can researchers troubleshoot common issues with EML4 antibody experiments?

When encountering problems with EML4 antibody experiments, researchers should consider these solution-oriented approaches:

Problem: Weak or No Signal in Western Blotting

• Solution 1: Optimize antibody concentration by testing a dilution series (1:500 to 1:3000) .

• Solution 2: Increase protein loading (30-50 μg total protein may be required).

• Solution 3: Extend primary antibody incubation time to overnight at 4°C.

• Solution 4: Ensure target protein is not degraded by including additional protease inhibitors in lysis buffer.

• Solution 5: Verify sample preparation method preserves native protein structure (particularly important for membrane and cytoskeletal proteins).

Problem: High Background in Immunohistochemistry

• Solution 1: Optimize antibody dilution (test range from 1:50 to 1:500) .

• Solution 2: Increase blocking time and/or concentration of blocking agent.

• Solution 3: Test different antigen retrieval methods (TE buffer pH 9.0 versus citrate buffer pH 6.0) .

• Solution 4: Include additional washing steps with PBS-T.

• Solution 5: Use alternative detection systems if current one shows high background.

Problem: Inconsistent Results Between Different Detection Methods

• Solution 1: Verify antibody specificity using CRISPR-Cas9 edited cell lines as negative controls .

• Solution 2: Use multiple antibody clones targeting different epitopes of EML4.

• Solution 3: Include positive control samples with confirmed EML4 expression (e.g., A549 cells) .

• Solution 4: Consider that heterogeneous expression may explain discrepancies between methods with different sensitivities .

• Solution 5: Document exact protocols, antibody lots, and experimental conditions to identify variables causing inconsistency.

Problem: Difficulty Detecting EML4-ALK Fusion Protein

• Solution 1: Use antibodies specifically validated for EML4-ALK fusion detection.

• Solution 2: Consider that >5% of cells must show cytoplasmic staining of at least grade 1 intensity for positive result interpretation .

• Solution 3: Verify results with alternative methods (FISH if using IHC, or vice versa).

• Solution 4: Use cell lines expressing specific EML4-ALK variants (e.g., H2228 expressing variant 3) as positive controls .

• Solution 5: Remember that transcript detection may not correlate with protein expression .

What considerations are important when selecting and validating EML4 antibodies for specific research applications?

Selecting and validating the appropriate EML4 antibody requires careful consideration of multiple factors:

• Target epitope location:

  • For detecting full-length EML4, antibodies targeting conserved regions are preferred

  • For EML4-ALK fusion protein detection, antibodies raised against epitopes near the fusion junction may provide specificity

  • Some antibodies are generated against N-terminal regions (e.g., residues surrounding Arg91 in human EML4) , while others target C-terminal regions (e.g., aa 900 to C-terminus)

• Antibody type:

  • Polyclonal antibodies offer detection of multiple epitopes, potentially increasing sensitivity

  • Monoclonal antibodies provide consistency between batches and potentially higher specificity

  • Consider that polyclonal preparations have limited supply and may vary between lots

• Validation methods:

  • Western blotting should show a band at approximately 120 kDa

  • CRISPR-Cas9 edited cell lines provide definitive negative controls

  • Cross-reactivity testing against related proteins (e.g., other EML family members) ensures specificity

• Application-specific validation:

  • For WB: Verify linear range of detection with protein dilution series

  • For IHC: Test on tissues with known EML4 expression profiles (e.g., breast, prostate, and lung cancer tissues)

  • For IF/ICC: Confirm localization pattern matches known EML4 cellular distribution

  • For IP: Validate recovery efficiency with subsequent Western blot analysis

• Documentation and reproducibility:

  • Check if the antibody has Registered Research Resource Identifier (RRID) for tracking literature usage

  • Review published literature using the specific antibody clone

  • Consider commercial antibodies with extensive validation data across multiple applications

By systematically addressing these considerations, researchers can select antibodies most likely to yield reliable, reproducible results for their specific experimental questions involving EML4.

How is EML4-ALK research advancing our understanding of drug resistance mechanisms in cancer?

Recent studies on EML4-ALK are providing critical insights into drug resistance mechanisms:

The formation of EML4-ALK cytoplasmic protein condensates represents a novel structural aspect of this fusion protein that impacts therapeutic response . These assemblies result from networks of interactions between oncogene and adapter protein multimers, creating potentially distinct microenvironments within cancer cells.

Cutting-edge research using optogenetics and live-cell imaging has revealed that EML4-ALK assemblies suppress transmembrane receptor tyrosine kinase (RTK) signaling . This finding is particularly significant because RTK activation represents a major bypass mechanism for resistance to ALK inhibitors in clinical settings. When ALK is inhibited by targeted therapies, cancer cells often adapt by activating alternative RTK pathways to maintain growth and survival signaling.

The physical properties of these protein assemblies may affect drug penetration, binding kinetics, and downstream signaling, all of which could contribute to variable therapeutic responses among patients. Understanding these biophysical characteristics opens new avenues for drug development strategies that might specifically target the assembly formation rather than just ALK kinase activity.

This evolving understanding of EML4-ALK's structural organization challenges previous models that focused primarily on kinase domain mutations as the predominant resistance mechanism. It suggests that spatial organization and protein-protein interaction networks play equally important roles in therapeutic response and resistance development.

Future therapeutic approaches may need to consider both inhibiting kinase activity and disrupting protein assemblies to achieve more durable responses in EML4-ALK-driven cancers.

What new experimental models and technologies are advancing EML4 and EML4-ALK research?

The field of EML4 and EML4-ALK research is being transformed by several innovative experimental models and technologies:

Optogenetic Systems:
Recent studies have employed optogenetics to manipulate EML4-ALK activity with precise temporal control . This approach allows researchers to induce or disrupt protein assemblies using light stimulation, enabling detailed investigation of the kinetics and reversibility of EML4-ALK signaling events. By coupling optogenetic manipulation with live-cell imaging, researchers can visualize real-time changes in protein localization and downstream signaling pathways.

CRISPR-Cas9 Genome Editing:
CRISPR-Cas9 technology has facilitated the creation of precisely edited cell lines that serve as powerful tools for antibody validation and functional studies . These include:

• Complete EML4 knockout cell lines for definitive negative controls

• Cell lines with specific domain deletions to study structure-function relationships

• Knock-in cell lines expressing tagged versions of EML4 for localization studies

Patient-Derived Organoids (PDOs):
Three-dimensional organoid cultures derived from patient samples provide more physiologically relevant models than traditional cell lines. PDOs maintain tumor heterogeneity and microenvironment components, allowing for more accurate assessment of drug responses and resistance mechanisms in EML4-ALK-positive cancers.

Proximity Labeling Techniques:
BioID and APEX2-based proximity labeling approaches are revealing the protein interaction networks surrounding EML4 and EML4-ALK fusion proteins. These techniques identify proteins that are physically close to the target protein in living cells, helping map the complex interactome that contributes to oncogenic signaling and drug resistance.

High-Resolution Imaging: Super-resolution microscopy techniques are providing unprecedented visualization of EML4-ALK assemblies at the nanoscale level. These approaches reveal the spatial organization of these protein condensates and their relationship to cellular structures like the cytoskeleton and membrane-bound organelles.