emc7 Antibody

What is EMC7 and what cellular functions has it been implicated in?

EMC7 is a subunit of the Endoplasmic Reticulum Membrane Protein Complex that plays essential roles in membrane biology. Research has demonstrated that EMC7 functions as a molecular tether between the late endosome (LE) and endoplasmic reticulum (ER), facilitating inter-organelle communication and cargo transport . This tethering occurs through EMC7's interaction with the LE-associated Rab7 GTPase, which stabilizes contact between these organelles .

Notably, EMC7 has been implicated in viral infection pathways, particularly for polyomaviruses like SV40. Studies show that EMC7 and EMC4 support SV40 infection by promoting LE-to-ER targeting of the virus, which is essential for productive infection . Additionally, Gene Ontology annotations suggest EMC7 may possess carbohydrate binding capabilities, though this requires further investigation .

What are the key structural features of EMC7 that researchers should consider when selecting antibodies?

EMC7 contains several important structural domains that researchers should consider when selecting antibodies for specific experiments:

• Cytosolic domains: EMC7 contains a cytosolic C-terminal domain with protein-protein interaction capabilities

• Disordered domain: Located at positions 188-201 within the C-terminal cytosolic domain

• Low Complexity Region (LCR): Found at positions 218-239 within the C-terminal cytosolic domain

• Transmembrane regions: EMC7 is an ER transmembrane protein with specific membrane topology

When selecting antibodies, researchers should consider which domain they wish to target. For instance, antibodies targeting the disordered domain or LCR may interfere with Rab7 binding, as truncated EMC7 constructs lacking these regions showed defects in binding to EGFP-Rab7 in experimental studies . Commercially available antibodies may recognize different epitopes - for example, one polyclonal antibody targets an immunogen sequence "DMRREMEQSM NMLNSNHELP DVSEFMTRLF SSKSSGKSSS GSSKTGKSGA GK" , while another targets "VVPGVKPQDWI SAARVLVDGE EHVGFLKTDG SFVVHDIPSG SYVVEVVSPA YRFDPVRVDI TSKGKMRARY VNYIKTSEVV RLPYPLQMKS SGPPSYFIKR ESWGW" .

What methodological considerations are important when designing experiments using EMC7 antibodies?

When designing experiments with EMC7 antibodies, consider these methodological factors:

• Application compatibility: Verify that your selected antibody has been validated for your specific application (WB, IP, IF, IHC, etc.). For example, some EMC7 antibodies are validated for Western Blot, IP, IF, and ELISA applications .

• Species reactivity: Confirm cross-reactivity with your experimental model. Many EMC7 antibodies react with human, mouse, and rat samples , but predicted reactivity may extend to other species like bovine, canine, and rabbit for some antibodies .

• Protocol optimization:

  • For Western blotting: recommended dilutions vary by manufacturer but typically range from 0.2-1 μg/mL

  • For immunofluorescence: antibodies should be tested at different concentrations with appropriate controls

  • For immunoprecipitation: conditions may need optimization, particularly when studying interactions with partners like Rab7

• Epitope accessibility: Consider whether the epitope will be accessible in your experimental conditions, especially when studying membrane-bound EMC7 in its native conformation .

How have researchers successfully employed EMC7 antibodies to study membrane contact sites?

Researchers have used several sophisticated approaches with EMC7 antibodies to investigate membrane contact sites:

• Co-immunoprecipitation assays: EMC7 antibodies have been used to precipitate the protein and identify binding partners. Studies showed that immunoprecipitation of EMC7 co-precipitated Rab7, demonstrating their interaction .

• BioID proximity labeling: Researchers fused BioID2 to EMC7 (EMC7-BioID2-HA) to identify proteins in close proximity. This approach showed that EMC7 was in close physical proximity to Rab7, supporting the concept that EMC7 participates in ER-LE contact sites .

• Super-resolution microscopy: Structured Illumination Microscopy (SIM) experiments revealed that colocalization between the ER and LE was disrupted in cells depleted of EMC7, providing visual evidence of EMC7's role in maintaining ER-LE contacts .

• Split-GFP approaches: This technique demonstrated that EMC7 promotes ER-LE contact formation .

• Immuno-electron microscopy: Researchers used EMC7 antibodies in immuno-EM to visualize EMC7 at ER-LE contact sites, providing ultrastructural evidence of its precise localization .

What controls are essential when performing immunodetection using EMC7 antibodies?

When using EMC7 antibodies, these controls are essential for rigorous experimental design:

• Negative controls:

  • Isotype controls: Use matched isotype antibodies of the same species to assess non-specific binding

  • Secondary-only controls: Omit primary antibody but include secondary antibody to evaluate background

  • Cells with EMC7 knockdown: Research has employed EMC7 siRNA, shRNA, or CRISPR/Cas9 knockout systems to generate negative control samples

• Positive controls:

  • Known EMC7-expressing tissues/cells: Human muscle has been used as a positive control for some EMC7 antibodies

  • Recombinant EMC7: Overexpressed tagged EMC7 can serve as a strong positive control

• Specificity controls:

  • Peptide competition assays: Pre-incubating the antibody with the immunizing peptide should abolish specific staining

  • Multiple antibodies to different epitopes: Using antibodies targeting different regions of EMC7 can help confirm specificity

How can EMC7 antibodies be used to investigate viral infection pathways?

EMC7 antibodies have been instrumental in elucidating viral infection mechanisms, particularly for polyomaviruses:

• Tracking virus-organelle interactions: Immuno-EM studies using EMC7 antibodies have visualized SV40 particles in Rab7-positive LEs making contact with EMC7-positive ER membranes, providing direct evidence of virus trafficking through these contact sites .

• Infection pathway analysis: By coupling EMC7 antibodies with viral infection assays, researchers determined that EMC7 depletion markedly reduced SV40 infection rates and impaired virus delivery to the ER .

• Membrane contact site characterization: EMC7 antibodies helped demonstrate that EMC7 forms a molecular bridge between the LE and ER by binding both Rab7 on the LE and syntaxin18 (Stx18) on the ER, facilitating viral transport between compartments .

• Structure-function analysis: By using EMC7 antibodies alongside truncated EMC7 mutants, researchers identified that the disordered domain and LCR within EMC7's cytosolic domain are critical for Rab7 binding and SV40 transport to the ER .

What methodological approaches can resolve contradictory results when using EMC7 antibodies?

When faced with contradictory results using EMC7 antibodies, consider these methodological approaches:

• Validate antibody specificity:

  • Test the antibody in EMC7 knockout/knockdown cells to confirm specificity

  • Use multiple antibodies targeting different epitopes to corroborate findings

  • Consider cross-reactivity with other EMC subunits, as the ER membrane complex contains multiple components

• Optimize experimental conditions:

  • Test different fixation methods for immunofluorescence (paraformaldehyde vs. methanol)

  • Vary antigen retrieval methods for immunohistochemistry

  • Adjust detergent concentrations when extracting membrane proteins

• Employ complementary techniques:

  • Combine antibody-based approaches with genetic tools (CRISPR, siRNA) to confirm findings

  • Use proximity labeling techniques like BioID alongside co-immunoprecipitation

  • Apply live-cell imaging approaches to complement fixed-cell observations

• Consider context-dependent factors:

  • Cell type differences in EMC7 expression or localization

  • Potential changes in EMC7 interactions under different physiological states

  • Post-translational modifications that might affect antibody recognition

How do the protein domains of EMC7 affect antibody selection for specific research questions?

The domain architecture of EMC7 significantly impacts antibody selection for different research questions:

For studying:

• Protein interactions: Select antibodies targeting regions outside the disordered domain and LCR to avoid disrupting native interactions

• Membrane localization: Choose antibodies recognizing cytosolic domains that remain accessible in intact cells

• Protein structure analyses: Consider antibodies targeting conserved regions if working across species

What are the advanced considerations for using EMC7 antibodies in studying membrane contact sites?

Advanced researchers investigating membrane contact sites should consider these sophisticated approaches with EMC7 antibodies:

• Super-resolution microscopy optimization:

  • When using structured illumination microscopy (SIM) to visualize EMC7 at ER-LE contacts, optimize fixation methods that preserve membrane architecture

  • Consider dual-color STORM or PALM imaging with EMC7 and Rab7 antibodies to achieve nanometer-scale resolution of contact sites

• Temporal dynamics analysis:

  • Use EMC7 antibodies in pulse-chase experiments to track the formation and dissolution of contact sites

  • Consider photoactivatable or photoconvertible EMC7 fusions combined with antibody labeling of interaction partners

• Quantitative contact site measurements:

  • Develop automated image analysis pipelines to quantify ER-LE contacts using EMC7 antibody labeling

  • Combine with split-GFP approaches to measure contact site formation kinetics

• Context-dependent contact site formation:

  • Investigate how viral infection, cellular stress, or signaling events affect EMC7 localization and contact site formation

  • Consider proteomic analysis of EMC7 immunoprecipitates under different conditions to identify context-specific interaction partners

• Structural organization of contact sites:

  • Use EMC7 antibodies with correlative light and electron microscopy (CLEM) to bridge fluorescence localization with ultrastructural details

  • Consider cryo-electron tomography to visualize native EMC7-containing contact sites at molecular resolution

How can researchers optimize co-immunoprecipitation experiments with EMC7 antibodies to study protein-protein interactions?

For optimizing co-immunoprecipitation (co-IP) experiments with EMC7 antibodies to study protein interactions:

• Membrane protein extraction optimization:

  • Test different detergents (digitonin, CHAPS, DDM) to solubilize EMC7 while preserving native interactions

  • Consider crosslinking approaches before lysis to stabilize transient interactions

• Binding partner considerations:

  • For studying EMC7-Rab7 interactions, use GTP-locked mutants (e.g., EGFP-Q67L Rab7) to maximize binding, as EMC7 preferentially binds the GTP-bound form of Rab7

  • Consider that EMC7 interacts with ER-resident syntaxin18 and that EMC7 depletion may affect this interaction

• Technical optimization:

  • Adjust antibody concentrations and incubation times to maximize specific pull-down

  • Consider using tagged versions of EMC7 (EMC7-FLAG) alongside antibodies for more efficient precipitation

  • Test both direct IP of EMC7 and reverse IP of binding partners like Rab7

• Controls for specificity:

  • Include IP with non-related antibodies as negative controls

  • Use EMC7-depleted cells as specificity controls

  • For Rab7 interaction studies, include non-binding mutants like EGFP-N125I Rab7, which exists in the apo form devoid of GTP or GDP

What approaches can differentiate between general EMC complex functions and EMC7-specific roles?

To distinguish EMC7-specific functions from general EMC complex activities:

• Selective subunit depletion:

  • Compare phenotypes of EMC7 knockdown with depletion of other EMC subunits (EMC1-10)

  • Research has shown that EMC4 and EMC7 have specific roles in SV40 viral trafficking, distinct from other subunits

• Domain-specific mutations:

  • Utilize truncated EMC7 constructs lacking specific domains (e.g., ΔD1 EMC7*-FLAG, ΔD2 EMC7*-FLAG, or ΔD1, D2 EMC7*-FLAG)

  • These constructs maintain association with other EMC subunits but lose specific functions like Rab7 binding

• Interaction partner analysis:

  • Compare interaction partners of different EMC subunits through parallel co-IP or BioID experiments

  • For example, EMC7 interacts with Rab7 and syntaxin18, which may be specific to this subunit

• Rescue experiments:

  • Perform complementation studies with wild-type vs. mutant EMC7 in EMC7-depleted cells

  • Research showed that wild-type EMC7*-FLAG rescued SV40 infection in EMC7-depleted cells, while the ΔD1, D2 EMC7*-FLAG mutant did not

How should researchers interpret EMC7 antibody results in disease-related studies?

When interpreting EMC7 antibody results in disease contexts:

• Disease association considerations:

  • EMC7 has been associated with Cone-Rod Dystrophy, X-Linked, 1

  • Consider potential changes in EMC7 expression, localization, or interactions in disease states

• Viral infection mechanism insights:

  • EMC7 plays a critical role in polyomavirus infection pathways, including BK polyomavirus (BK PyV), JC polyomavirus (JC PyV), and SV40

  • Changes in EMC7 levels or function may impact viral pathogenesis

• Tissue-specific expression patterns:

  • Use EMC7 antibodies to examine expression patterns across tissues in normal vs. disease states

  • Consider how tissue-specific post-translational modifications might affect antibody recognition

• Organelle communication defects:

  • As EMC7 functions in ER-LE communication, examine how this process might be disrupted in diseases affecting membrane trafficking

  • Look for colocalization changes between EMC7 and Rab7 or syntaxin18 in disease models

• Therapeutic implications:

  • Consider how findings regarding EMC7's role in viral infection might inform therapeutic approaches

  • For viral diseases, EMC7 might represent a potential target for disrupting viral trafficking

What are the advantages and limitations of different types of EMC7 antibodies for specific research applications?

Understanding the comparative advantages of different EMC7 antibody types is essential for experimental design:

For challenging applications:

• Membrane contact site visualization: Super-resolution microscopy often works better with monoclonal antibodies due to their precision

• Co-IP of membrane complexes: Polyclonal antibodies may provide better pull-down efficiency

• Multiple labeling experiments: Consider using antibodies from different host species to allow simultaneous detection

How can researchers employ EMC7 antibodies alongside genetic manipulation techniques?

Integrating EMC7 antibodies with genetic tools creates powerful experimental approaches:

• Knockdown/knockout validation:

  • Use EMC7 antibodies to confirm protein reduction in EMC7 siRNA , shRNA , or CRISPR/Cas9 knockout experiments

  • Researchers have confirmed EMC7 depletion using antibodies following siRNA treatment

• Rescue experiments:

  • After EMC7 knockdown, express modified EMC7 (resistant to siRNA) and use antibodies to confirm expression

  • Studies have used EMC7*-FLAG constructs in rescue experiments and confirmed expression and localization using antibodies

• Domain function analysis:

  • Express truncated EMC7 constructs (ΔD1 EMC7*-FLAG, ΔD2 EMC7*-FLAG) and use antibodies to assess interaction partner binding

  • Research showed these constructs maintain EMC complex incorporation but lose Rab7 binding

• Proximity labeling approaches:

  • Combine BioID-tagged EMC7 (EMC7-BioID2-HA) with antibodies against potential interaction partners

  • This approach identified Rab7 as an EMC7 proximity partner

• CRISPR activation systems:

  • Use EMC7 CRISPR activation plasmids to upregulate expression and confirm with antibodies

  • This approach can help establish gain-of-function phenotypes

What are the latest methodological advances in studying EMC7 using antibody-based approaches?

Recent methodological advances for EMC7 research using antibodies include:

• Proximity-dependent labeling technologies:

  • BioID approaches have been successfully applied to EMC7, fusing BioID2 to the cytosolic C-terminus (EMC7-BioID2-HA) to identify proteins in close proximity

  • This approach can identify weak or transient interactions that might be missed by traditional co-IP

• Super-resolution microscopy applications:

  • Structured Illumination Microscopy (SIM) has been used to visualize EMC7-dependent colocalization between ER and LE

  • These techniques overcome diffraction limits to provide nanoscale resolution of membrane contact sites

• Split-fluorescent protein approaches:

  • Split-GFP systems allow direct visualization of protein proximity in living cells

  • This approach demonstrated that EMC7 promotes ER-LE contact formation

• Correlative microscopy techniques:

  • Combining light microscopy (using EMC7 antibodies) with electron microscopy offers complementary insights

  • Immuno-EM has visualized EMC7 at ER-LE contact sites during SV40 trafficking

• Quantitative proteomic analysis:

  • Using EMC7 antibodies for immunoprecipitation followed by mass spectrometry enables comprehensive interaction partner identification

  • This approach can be combined with SILAC or TMT labeling for quantitative comparison between conditions