EML1 Antibody

What is EML1 and why is it an important research target?

EML1 (Echinoderm Microtubule Associated Protein Like 1) is a protein that modulates the assembly and organization of the microtubule cytoskeleton, playing a crucial role in regulating mitotic spindle orientation and the orientation of the plane of cell division . It is required for normal proliferation of neuronal progenitor cells in the developing brain and for normal brain development . Research has also demonstrated EML1's importance in retinal photoreceptor migration and survival , and its essential role in oocyte meiotic maturation . EML1 mutations have been associated with subcortical heterotopia in the brain, hydrocephalus, and cognitive impairment , making it a significant target for neurodevelopmental research.

What types of EML1 antibodies are available for research applications?

EML1 antibodies are available in various formats that suit different research applications:

These antibodies target different epitopes of EML1, with some specifically recognizing the C-terminal region (aa 550 to C-terminus) or central regions (aa 100-200) of the protein .

What are the most validated applications for EML1 antibodies?

According to published literature and antibody validation data, EML1 antibodies have been successfully used in multiple applications:

• Western Blot (WB): Most validated application, with dilution ranges typically from 1:500 to 1:6000

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Successfully used to visualize EML1 localization in cells, particularly on spindle structures during cell division

• Immunohistochemistry (IHC): Validated for tissue sections, especially for brain and retinal tissues

• ELISA: Used in some antibody formulations, primarily for protein quantification

The specific application determines which antibody format is most appropriate, with polyclonal antibodies generally offering broader reactivity and monoclonal antibodies providing higher specificity .

How should I optimize Western blot protocols for detecting EML1 protein?

For optimal Western blot detection of EML1:

• Sample preparation: Use appropriate lysis buffers containing protease inhibitors to prevent degradation of EML1 (molecular weight ~90 kDa)

• Gel selection: Use 7.5% SDS-PAGE gels which provide better resolution for the EML1 protein (observed MW: 90-92 kDa)

• Transfer conditions: Optimize for larger proteins using lower voltage for longer time or semi-dry transfer systems

• Antibody dilution: Start with manufacturer's recommended dilution (typically 1:1000-1:6000 for WB) and optimize based on signal-to-noise ratio

• Detection system: Use enhanced chemiluminescence systems for sensitive detection

• Controls: Include positive controls such as brain tissue lysates from mouse or rat, which show high expression of EML1

• Loading control: Use β-actin as a validated loading control as shown in published EML1 research

Note that some commercial antibodies may only recognize specific isoforms of EML1. For example, one study reported that a polyclonal antibody (PA5-30016) recognized only the short EML1 isoform (~85-89 kDa) but not the long isoform .

What are the recommended protocols for immunofluorescence staining using EML1 antibodies?

For successful immunofluorescence staining of EML1:

• Fixation method: Use paraformaldehyde (PFA) fixation (typically 4%) with Triton X-100 permeabilization for cultured cells

• Blocking: Block with 3-5% BSA or normal serum (from the species of secondary antibody) for 1 hour at room temperature

• Primary antibody: Apply EML1 antibody at appropriate dilution (typically 1:10-1:100 for IF/ICC) and incubate overnight at 4°C

• Secondary antibody: Use fluorescently labeled secondary antibodies specific to the host species of the primary antibody

• Nuclear counterstaining: Include DAPI or other nuclear stains to visualize nuclei

• Mounting: Use anti-fade mounting medium to preserve fluorescence

• Controls: Include both positive controls (tissues/cells known to express EML1) and negative controls (primary antibody omission)

• Colocalization studies: Consider double-staining with microtubule markers (e.g., α-tubulin) to visualize EML1's association with microtubule networks

For specific applications like visualizing EML1 on the meiotic spindle in oocytes, specialized protocols have been published that demonstrate colocalization with other proteins like NUDC .

How can I validate the specificity of EML1 antibodies in my experimental system?

Validating antibody specificity is crucial for reliable results. For EML1 antibodies, consider these approaches:

• Knockdown/knockout validation:

  • Use siRNA, shRNA, or morpholino oligos to reduce EML1 expression (as demonstrated in oocyte studies)

  • Compare antibody signal in control vs. knockdown samples by Western blot and immunostaining

  • Effective knockdown should show proportional reduction in signal intensity (e.g., 57.1% reduction was observed in morpholino studies)

• Overexpression validation:

  • Express tagged EML1 (e.g., DDK/FLAG-tagged EML1) and detect with both anti-EML1 and anti-tag antibodies

  • Overlapping signals confirm antibody specificity

  • This approach has been validated in both HEK293 cells and oocytes

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide before application

  • Signal should be significantly reduced or eliminated

• Multiple antibody validation:

  • Use different antibodies targeting different epitopes of EML1

  • Similar patterns support specificity

• Expected subcellular localization:

  • Confirm localization patterns match expected biology (e.g., microtubule association, spindle localization)

How does EML1 localization change during cell division, and how can this be effectively studied with antibodies?

EML1 demonstrates dynamic localization patterns during cell division that can be visualized using immunofluorescence techniques:

• Cell cycle-dependent localization:

  • EML1 associates with the microtubule network in interphase cells

  • During mitosis, it localizes to the mitotic spindle

  • In oocyte meiosis, EML1 shows strong localization to meiotic spindles at metaphase I and II stages

• Experimental approach:

  • Synchronize cells at different cell cycle stages using cell cycle inhibitors

  • Fix cells and perform double immunostaining for EML1 and tubulin

  • Include cell cycle markers (e.g., phospho-histone H3 for mitosis)

  • Use confocal microscopy for high-resolution imaging

• Drug perturbation experiments:

  • Test dependence on microtubule integrity using depolymerizing agents (nocodazole) and stabilizing agents (taxol)

  • Studies have shown that nocodazole treatment causes loss of EML1 spindle localization, while taxol treatment maintains EML1 on stabilized spindles

• Live-cell imaging:

  • Express fluorescently tagged EML1 (e.g., GFP-EML1 or EML1-DDK) for live-cell imaging

  • Track dynamic changes throughout the cell cycle

  • Validate that tagged protein shows same localization as endogenous protein by fixed-cell immunofluorescence

These approaches have revealed EML1's critical role in spindle organization and proper chromosome segregation.

What approaches can be used to study EML1 protein interactions in cellular contexts?

To investigate EML1 protein interactions and complexes:

• Co-immunoprecipitation (Co-IP):

  • Use anti-EML1 antibodies for immunoprecipitation followed by Western blot detection of potential interacting partners

  • Alternatively, use tagged EML1 constructs (e.g., DDK/FLAG-tagged) for pulldown with anti-tag antibodies

  • This approach successfully identified NUDC as an EML1 interaction partner

• Mass spectrometry-based approaches:

  • Perform immunoprecipitation of EML1 followed by mass spectrometry analysis

  • This approach identified 514 potential EML1-interacting proteins, including tubulin subunits, actin regulation proteins, and other EML family members

• Proximity labeling methods:

  • Express EML1 fused to BioID or APEX2 to label proximal proteins in living cells

  • Identify interaction partners by streptavidin pulldown and mass spectrometry

• Immunofluorescence co-localization:

  • Perform double immunostaining for EML1 and potential interacting partners

  • Analyze co-localization by confocal microscopy

  • Verify using super-resolution microscopy techniques

  • This approach confirmed EML1 and NUDC co-localization on meiotic spindles

• Functional validation:

  • Use knockdown/knockout approaches to deplete EML1

  • Examine effects on localization and function of interaction partners

  • For example, EML1 knockdown caused displacement of NUDC from spindles

Gene enrichment analysis of EML1 interactors has revealed its involvement in multiple cellular processes including "RNA metabolism/splicing/localization," "cytoplasmic ribosomal subunit composition," and "protein folding and translation" .

How can researchers address the challenge of EML1 isoform specificity in their experiments?

EML1 has multiple isoforms, which presents challenges for antibody-based studies. To address this:

• Isoform characterization:

  • Determine which EML1 isoforms are expressed in your experimental system using RT-PCR with isoform-specific primers

  • Western blot analysis can identify the molecular weights of expressed isoforms (typically ~85-90 kDa)

• Antibody selection:

  • Choose antibodies that recognize epitopes common to all isoforms for total EML1 detection

  • Select epitope-specific antibodies for studying particular isoforms

  • Be aware that some antibodies may preferentially detect certain isoforms (e.g., some commercial antibodies recognize only the short EML1 isoform)

• Validation strategies:

  • Express recombinant isoforms and test antibody reactivity

  • Use tissues known to express specific isoforms as positive controls

  • Consider using tagged constructs of specific isoforms for overexpression studies

• Genetic approaches:

  • Design isoform-specific knockdown/knockout strategies

  • Use precise gene editing techniques like CRISPR/Cas9 to target specific isoforms

  • When studying EML1 mutants, consider which isoforms are affected by the mutation

• Interpreting contradictory results:

  • When results between studies differ, consider whether isoform specificity could explain the discrepancy

  • Compare the antibodies used and their known isoform recognition patterns

  • Some research has shown differences between in vivo mutant phenotypes and in vitro knockdown effects, possibly due to compensatory mechanisms involving other EML family members

What are the appropriate models for studying EML1 function in neurodevelopment?

Several models have been established for investigating EML1's role in neurodevelopment:

• Mouse models:

  • The Eml1 tvrm360 mutant mouse exhibits subcortical heterotopia in the brain with associated hydrocephalus and cognitive impairment

  • This model is valuable for studying EML1's role in neuronal progenitor proliferation and migration

  • Limited availability of viable mutant mice may necessitate complementary approaches

• In vitro neuronal cultures:

  • Primary neuronal cultures from wild-type and EML1 mutant animals

  • Neural progenitor cell cultures to study proliferation effects

  • Neurosphere assays to assess stem cell properties

• Knockdown approaches:

  • Morpholino or siRNA-based knockdown in relevant cell types

  • In utero electroporation for spatiotemporally controlled knockdown in developing brain

• Human cellular models:

  • iPSC-derived neural progenitors and organoids

  • CRISPR/Cas9-engineered cell lines with EML1 mutations

  • Patient-derived cells with naturally occurring EML1 mutations

• Imaging techniques:

  • Immunohistochemistry with EML1 antibodies on brain sections

  • Live imaging of fluorescently labeled neural progenitors

  • Time-lapse microscopy to track migration and division orientation

When interpreting results, consider that phenotypic differences between models may arise from experimental context (acute vs. chronic loss) or compensatory mechanisms involving other EML family members .

How can EML1 antibodies be used in studies of retinal development and disease?

EML1 antibodies have proven valuable in retinal research:

• Expression pattern analysis:

  • Immunohistochemistry using anti-EML1 antibodies to map expression across retinal layers

  • Western blot analysis to quantify expression during development or in disease models

• Functional studies:

  • Identification of mislocalized photoreceptors in Eml1 mutant retinas

  • Analysis of photoreceptor migration defects via immunostaining

  • Correlation of structural changes with functional impairments in ERG recordings

• Protein interaction studies:

  • Co-immunoprecipitation to identify retina-specific EML1 interaction partners

  • Co-localization studies with phototransduction proteins

• Disease models:

  • Eml1 mutant mice show reduction in scotopic light response and mislocalized photoreceptors

  • Potential relevance to Usher syndrome type 1A, as EML1 has been identified as a candidate gene

  • Western blot analysis revealed reduced expression of phototransduction proteins (rhodopsin, transducin subunits, PDEγ) in Eml1 mutants

• Technical considerations:

  • For retinal sections, specific fixation and antigen retrieval protocols may be necessary

  • When analyzing protein expression changes, β-actin has been validated as an appropriate loading control

  • Consider using multiple antibodies targeting different EML1 epitopes to ensure comprehensive detection

Research has shown that while EML1 is essential for photoreceptor migration and survival, it does not modulate phototransduction in mature rods and cones .

What methodological considerations should be addressed when using EML1 antibodies in immunogenicity studies?

When EML1 antibodies are used as part of immunogenicity studies or when studying anti-drug antibodies:

• Testing scheme design:

  • Implement a multi-tiered testing approach similar to standard anti-drug antibody (ADA) testing

  • Include screening assay, confirmation assay, and if positive, titer determination

  • For therapeutic antibodies, also include neutralizing antibody (NAb) assays

• Data structure and analysis:

  • Organize data following standardized formats such as SDTM IS (Immunogenicity Specimen Assessments) domain

  • Derive appropriate variables at analysis level to track treatment-emergent responses

  • Consider both binding antibodies and neutralizing antibodies in analysis

• Controls and validation:

  • Include appropriate positive and negative controls

  • Perform assay validation including determination of cut-points

  • Assess potential drug interference

• Interpretation challenges:

  • Distinguish between pre-existing and treatment-emergent antibodies

  • Evaluate persistence of antibody responses over time

  • Correlate antibody development with PK/PD parameters and clinical outcomes

  • Consider that anti-drug antibodies can impact drug concentration and efficacy measurements

• Statistical considerations:

  • Calculate incidence rates with appropriate denominators

  • Apply statistical methods for evaluating impact on efficacy and safety

  • Account for potential confounding factors

This approach enables proper evaluation of immunogenicity risk and definition of appropriate risk mitigation strategies .

How can fragment-based computational design approaches be applied to developing new EML1 antibodies?

Recent advances in computational antibody design offer promising approaches for developing novel EML1-targeting antibodies:

• Epitope selection and analysis:

  • Identify accessible epitopes on EML1 structure using computational surface analysis

  • Target functionally important regions based on known biology

  • Consider epitope conservation across species if cross-reactivity is desired

• Fragment-based design approach:

  • Identify protein fragments that can interact with selected epitopes

  • Combine interacting fragments to design complementarity-determining regions (CDRs)

  • Graft designed CDRs onto antibody scaffolds using structural matching or direct grafting approaches

• Computational optimization:

  • Optimize side-chain interactions between designed antibody and EML1 epitope

  • Enhance solubility and conformational stability through computational refinement

  • Predict binding affinity using molecular dynamics simulations

• Experimental validation pipeline:

  • Express designed antibodies in prokaryotic systems (e.g., E. coli)

  • Perform biophysical characterization (circular dichroism, thermal stability)

  • Test binding using ELISA, surface plasmon resonance, or other binding assays

  • Validate functionality in cellular assays

• Advantages of this approach:

  • Enables precise targeting of predetermined epitopes

  • Reduces time and cost compared to traditional antibody discovery methods

  • Works even with lower resolution structural data or computational models

  • Can generate antibodies with nanomolar binding affinities without in vitro affinity maturation

Fragment-based computational design has been successfully applied to generate antibodies against multiple targets, including the SARS-CoV-2 spike protein, and could be adapted for EML1-targeting applications .

What are common challenges when working with EML1 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with EML1 antibodies:

Additionally, when validating novel antibodies:

• Compare staining patterns with published localization data (e.g., spindle localization during cell division)

• Verify specificity through genetic approaches (knockdown/knockout)

• Consider using tagged EML1 constructs as positive controls

How should researchers integrate EML1 antibody data with other techniques to build comprehensive experimental evidence?

For robust research outcomes, integrate EML1 antibody-based techniques with complementary approaches:

• Multi-level protein analysis:

  • Combine Western blot (protein levels) with immunofluorescence (localization)

  • Supplement with mass spectrometry for unbiased protein identification and interactions

  • Use proximity labeling methods to identify proteins in the same cellular compartment

• Functional validation:

  • Correlate antibody-detected protein changes with functional outcomes

  • For example, link EML1 spindle localization with cell division phenotypes

  • Connect photoreceptor mislocalization with retinal response deficits

• Genetic approaches:

  • Use gene knockout/knockdown models to validate antibody specificity

  • Apply rescue experiments with wild-type or mutant constructs

  • EML1 knockdown studies have revealed its role in oocyte meiotic progression

• Live-cell imaging:

  • Complement fixed-cell antibody staining with live imaging of fluorescently tagged proteins

  • Track dynamic changes that might be missed in fixed samples

  • Validate that tagged protein behaves similarly to endogenous protein

• Multi-omics integration:

  • Correlate protein-level findings with transcriptomic data

  • Consider epigenetic regulation

  • Integrate with interactome data from immunoprecipitation-mass spectrometry studies

• Quantitative analysis:

  • Apply image analysis software for quantification of immunofluorescence signals

  • Use quantitative Western blot techniques with proper controls

  • Implement statistical analysis for rigorous interpretation