emc10 Antibody

What is EMC10 and why is it important in biological research?

EMC10 (ER membrane protein complex subunit 10) is a protein component of the endoplasmic reticulum membrane complex that facilitates energy-independent insertion of newly synthesized membrane proteins into ER membranes. With a canonical length of 262 amino acids and a mass of 27.3 kDa, EMC10 exists in multiple forms - a membrane-bound form (mEMC10) and a secreted form (scEMC10). Its importance spans multiple biological processes including neurodevelopment, thermogenesis regulation, sperm motility, and angiogenesis, making it a significant target for research across multiple disease states .

How do I select the appropriate EMC10 antibody for my experiment?

Selection depends on your experimental goals:

• For protein localization studies in tissue, choose antibodies validated for immunohistochemistry (IHC) or immunofluorescence (IF)

• For quantification studies, select antibodies optimized for Western blot or ELISA

• Consider antibody clonality: polyclonal antibodies offer higher sensitivity but potentially lower specificity than monoclonals

• Target epitope matters: N-terminal antibodies (AA 19-48) detect both membrane-bound and secreted forms, while some C-terminal antibodies may be specific to certain isoforms

• Verify species reactivity matches your experimental model (human, mouse, etc.)

What are the differences between membrane-bound EMC10 (mEMC10) and secreted EMC10 (scEMC10)?

These two forms have distinct functions and localization:

• mEMC10: Localized to the ER membrane as part of the EMC complex; involved in maintaining sodium homeostasis through regulation of Na+/K+-ATPase in sperm; crucial for sperm motility

• scEMC10: Secreted into circulation; functions as a signaling molecule that suppresses thermogenesis in brown adipose tissue; elevated in obesity and insulin resistance

• When designing experiments, consider that some antibodies may detect both forms while others might be specific to one form depending on their epitope targets

What are the optimal conditions for using EMC10 antibodies in Western blotting?

For optimal Western blot results with EMC10 antibodies:

• Recommended dilutions range from 1:500-1:2000, with many manufacturers suggesting 0.04-0.4 μg/mL as optimal concentration

• Use PVDF membranes for better protein retention

• Include proper positive controls (tissues with known EMC10 expression like testis or brain)

• For detecting scEMC10 in serum or culture media, immunoprecipitation prior to Western blotting may enhance sensitivity

• Blocking with 5% non-fat milk in TBST for 1 hour at room temperature works well for most EMC10 antibodies

• Primary antibody incubation is typically overnight at 4°C

• When probing for both mEMC10 and scEMC10, use size markers to distinguish between the full-length and processed forms

How can I optimize immunohistochemistry protocols for EMC10 detection in tissue samples?

For effective IHC with EMC10 antibodies:

• Antigen retrieval is critical - heat-induced epitope retrieval using citrate buffer (pH 6.0) is recommended

• Recommended dilutions typically range from 1:50-1:200

• For brain tissue, perfusion fixation provides superior results compared to immersion fixation

• EMC10 shows colocalization with MAP2 and NeuN in neuronal tissue, which can serve as positive controls

• For dual labeling, consider using antibodies raised in different host species to avoid cross-reactivity

• Blocking endogenous peroxidase activity is crucial when using HRP-conjugated detection systems

• Include negative controls (primary antibody omission and isotype controls) to validate specificity

What methodologies are available for detecting circulating scEMC10 in blood or serum samples?

Several approaches are validated for scEMC10 detection in circulation:

• ELISA: Commercial kits are available, or custom sandwich ELISA can be developed using capture and detection antibodies against different EMC10 epitopes

• Western blotting: May require sample concentration via immunoprecipitation prior to running

• Chemiluminescent immunoassay (CLIA): Has been successfully used for seminal plasma scEMC10 quantification

• Mass spectrometry: For absolute quantification when very high specificity is required

• For longitudinal studies tracking scEMC10 levels, consistent sampling conditions are crucial as levels may fluctuate with metabolic state

How can EMC10 antibodies be used to investigate the role of EMC10 in neurodevelopmental disorders?

Research on EMC10's role in neurodevelopmental contexts requires specialized approaches:

• For 22q11.2 deletion syndrome studies, compare EMC10 expression between patient-derived iPSC neurons and controls using quantitative immunofluorescence

• Perform co-localization studies with neuronal markers (MAP2, NeuN) to assess EMC10 expression patterns in specific neuronal populations

• Use EMC10 antibodies in combination with dendritic markers to analyze morphological changes in neurite outgrowth models

• For calcium signaling studies in neurons, combine EMC10 immunostaining with functional calcium imaging

• When investigating EMC10 knockdown effects, antibody validation becomes crucial to confirm reduction in protein levels following antisense oligonucleotide treatment or genetic manipulation

What experimental approaches can be used to study the role of scEMC10 in thermogenesis and metabolic regulation?

To investigate scEMC10's metabolic functions:

• Implement tissue-specific immunohistochemistry to analyze EMC10 expression in brown/white adipose tissues under different thermal conditions

• Use Western blot analysis of tissue lysates and serum to correlate tissue expression with circulating levels

• Employ neutralizing antibodies against scEMC10 for in vivo metabolic studies, monitoring parameters like energy expenditure and glucose homeostasis

• Combine EMC10 antibody staining with markers of thermogenic activation (UCP1, PGC1α) in adipose tissue sections

• For mechanistic studies, examine PKA-CREB/MAPK signaling pathway components following scEMC10 treatment using phospho-specific antibodies alongside EMC10 detection

How can I use EMC10 antibodies to investigate its role in cardiovascular pathologies?

For cardiovascular research applications:

• Perform immunohistochemistry on infarcted heart tissue sections to map EMC10 expression patterns in different cardiac zones (infarct core, border zone, remote myocardium)

• Use flow cytometry with EMC10 antibodies to identify and isolate EMC10-expressing cell populations from heart tissue

• Implement co-staining protocols with macrophage/monocyte markers to confirm bone marrow-derived cells as sources of EMC10 in infarcted hearts

• Quantify circulating scEMC10 levels using ELISA in correlation with cardiac function parameters

• For angiogenesis studies, combine EMC10 immunostaining with endothelial markers to assess capillary density in border zones of infarction

What are common issues when working with EMC10 antibodies and how can they be resolved?

Common challenges and solutions include:

• Weak signal: Try increasing antibody concentration, extending incubation time, using more sensitive detection systems, or enhancing antigen retrieval methods

• High background: Implement more stringent washing protocols, optimize blocking conditions (try different blockers like BSA instead of milk), or reduce secondary antibody concentration

• Multiple bands in Western blot: This may represent different EMC10 isoforms, processed forms, or post-translational modifications rather than non-specificity

• Inconsistent results between samples: Standardize sample preparation methods and ensure consistent protein loading

• Cross-reactivity: Validate specificity using EMC10 knockout/knockdown controls or peptide competition assays

How do I validate the specificity of an EMC10 antibody for my experimental system?

Rigorous validation approaches include:

• Genetic controls: Test antibody on samples from EMC10 knockout models or siRNA-mediated knockdown cells

• Peptide competition assays: Pre-incubate antibody with immunizing peptide to confirm binding specificity

• Multiple antibody comparison: Use antibodies targeting different epitopes of EMC10 to confirm consistent patterns

• Correlation of protein with mRNA expression: Compare antibody staining patterns with EMC10 mRNA distribution

• Western blot molecular weight verification: Confirm band appears at expected size (approximately 27.3 kDa for full-length protein)

• For recombinant expression systems, include tagged constructs (V5-tagged EMC10) that can be detected with both anti-EMC10 and anti-tag antibodies

How should I interpret discrepancies between EMC10 antibody results and functional data?

When facing contradictory results:

• Consider isoform specificity: Determine if your antibody detects all known EMC10 isoforms or is specific to particular forms

• Evaluate post-translational modifications: Some antibodies may not detect modified forms of EMC10

• Assess protein stability and turnover: The frameshift variant EMC10 287delG protein is rapidly degraded by the proteasome, making detection challenging despite RNA presence

• Examine subcellular localization: Membrane-bound versus secreted EMC10 may yield different results with certain antibodies

• Consider protein-protein interactions: EMC10 interactions with other proteins may mask epitopes in certain contexts

• Implement orthogonal methods: Combine antibody-based detection with mass spectrometry or functional assays to resolve discrepancies

How can EMC10 antibodies be utilized to explore its role in the ER membrane protein complex?

To investigate EMC10's role within the broader EMC complex:

• Implement co-immunoprecipitation with EMC10 antibodies followed by mass spectrometry to identify interaction partners

• Use proximity labeling approaches (BioID or APEX) with EMC10 antibodies to capture transient interactions

• Perform structured illumination or super-resolution microscopy with EMC10 antibodies to visualize its spatial organization within the ER membrane

• Combine EMC10 immunostaining with other EMC component antibodies to assess co-localization and complex integrity

• Use EMC10 antibodies in cell fractionation studies to determine the proportion of EMC10 integrated into the complex versus free forms

What considerations are important when using EMC10 antibodies to study its genetic variants in patient samples?

For genetic variant studies:

How can multiplexed antibody approaches advance our understanding of EMC10 in complex biological systems?

Multiplexed strategies offer powerful insights:

• Implement mass cytometry (CyTOF) with EMC10 antibodies alongside markers for cell type, signaling pathways, and metabolic state

• Use multiplex immunofluorescence with spectral unmixing to analyze EMC10 co-expression with up to 8-10 other proteins in the same tissue section

• Apply spatial transcriptomics combined with EMC10 immunohistochemistry to correlate protein expression with transcriptional profiles in tissue microenvironments

• Develop EMC10-targeted proximity ligation assays to visualize and quantify protein-protein interactions in situ

• For systems biology approaches, combine antibody-based proteomics with transcriptomics and metabolomics to build comprehensive models of EMC10 function in different physiological states