TMED10 Antibody

What is TMED10 and why is it important in research?

TMED10 (Transmembrane Emp24-Like Trafficking Protein 10) is a 25 kDa protein belonging to the EMP24/GP25L/p24 family. It functions as a cargo receptor involved in vesicular protein trafficking between the endoplasmic reticulum and Golgi apparatus . TMED10 is also known as TMP21, p23, and p24delta.

The protein has gained significant research interest due to its:

• Role in modulating gamma-secretase activity without affecting epsilon-secretase activity in Alzheimer's disease pathology

• Function in protein trafficking pathways, particularly for growth factors like IGF2

• Regulatory role in autophagy through interaction with ATG4B

• Involvement in TGF-β signaling through interference with receptor complex formation

• Potential role in T cell function through regulating PD-1 surface expression

What applications are TMED10 antibodies suitable for?

Based on validation data from multiple sources, TMED10 antibodies have demonstrated effectiveness in the following applications:

What is the expected molecular weight of TMED10 in western blot analysis?

While the calculated molecular weight of TMED10 is approximately 25 kDa, many researchers observe a band at approximately 21-23 kDa in western blot analysis . This discrepancy is likely due to post-translational modifications or proteolytic processing.

When using antibodies against different epitopes, you may observe:

• Antibodies targeting the center region typically detect a band at ~21-23 kDa

• Antibodies against the C-terminus may detect additional bands in some cell types

• Some commercially available antibodies report detecting bands between 20-25 kDa depending on the cell/tissue type

If multiple bands are observed, validation experiments including knockout/knockdown controls are recommended to confirm specificity.

Which species do TMED10 antibodies typically react with?

Most commercially available TMED10 antibodies demonstrate reactivity with:

How can I validate the specificity of my TMED10 antibody?

Validating antibody specificity is crucial for reliable experimental results. For TMED10 antibodies, consider these approaches:

• Genetic validation:

  • Test the antibody in TMED10 knockout/knockdown models

  • Several studies have successfully used siRNA against TMED10 to validate antibody specificity

  • CRISPR/Cas9-mediated knockout of TMED10 has been reported in HeLa cells and can be used as a negative control

• Peptide competition assay:

  • Pre-incubate the antibody with the immunogen peptide (many manufacturers offer blocking peptides)

  • The specific signal should be significantly reduced or eliminated

• Multiple antibody validation:

  • Use antibodies targeting different epitopes of TMED10

  • Consistent results across antibodies increase confidence in specificity

• Expression validation:

  • Compare antibody detection with TMED10 overexpression systems

  • Ectopic expression of TMED10 in experimental models should show increased signal

• Mass spectrometry validation:

  • Immunoprecipitate TMED10 and confirm identity by mass spectrometry

  • This approach has been used to validate TMED10 antibodies in protein interaction studies

What methodological approaches can I use to study TMED10's role in protein trafficking?

TMED10 functions as a cargo receptor in vesicular trafficking. To study this role, consider these methodologies:

• RUSH system (Retention Using Selective Hooks):

  • This approach has been successfully used to study TMED10-mediated IGF2 trafficking

  • In RUSH-IGF2-HA experiments, TMED10 knockdown significantly decreased IGF2 secretion efficiency

  • Control experiments showed this effect was specific to IGF2, as ShhN secretion was unaffected

• Subcellular fractionation and localization:

  • Use ultracentrifugation to isolate ER, Golgi, and vesicular fractions

  • Immunoblot with TMED10 antibodies to track its distribution

  • Combine with client protein detection to study co-trafficking

• Live-cell imaging with fluorescently tagged TMED10:

  • Co-express with tagged cargo proteins to visualize trafficking dynamics

  • TMED10-GFP fusion proteins have been used to study its localization and trafficking function

• Secretion assays:

  • Quantify secreted proteins in control versus TMED10-depleted cells

  • Mass spectrometry analysis of conditioned media from TMED10 knockdown cells identified multiple affected secretory proteins, including IGF2

• Biotinylation assays for cell surface proteins:

  • Label intact cells with non-membrane permeable biotinylation reagent

  • IP biotinylated proteins and immunoblot for TMED10

  • This approach has confirmed that a fraction of TMED10 localizes to the cell surface

How can I use TMED10 antibodies to study protein-protein interactions?

TMED10 interacts with various proteins in different cellular contexts. To study these interactions:

• Co-immunoprecipitation (Co-IP):

  • IP with TMED10 antibodies followed by immunoblotting for potential interaction partners

  • This approach has identified interactions between TMED10 and:

    • TGF-β receptors (ALK5 and TβRII)

    • ATG4B (autophagy-related protein)

    • PD-1 (immune checkpoint protein)

    • Other TMED family proteins

• Bimolecular Fluorescence Complementation (BiFC):

  • Fuse TMED10 and putative interacting proteins to complementary fragments of fluorescent proteins

  • This technique successfully visualized TMED10-ATG4B interactions in living cells

  • The interaction between TMED10-VN and ATG4B-VC generated strong fluorescence, validating direct protein interaction

• Proximity Ligation Assay (PLA):

  • Detect protein interactions at endogenous levels in fixed cells

  • Provides spatial information about where interactions occur within cells

• Affinity labeling experiments:

  • Used successfully to study TMED10 interactions with TGF-β receptors

  • For example, 125I-TGF-β labeling of receptors followed by IP with TMED10 antibodies

• Mapping interaction domains:

  • Generate deletion mutants of TMED10 to identify binding regions

  • Studies have shown that:

    • The lumenal domain (LD) of TMED10 is critical for ATG4B interaction

    • The region between Ile91 and Glu110 is crucial for TGF-β receptor binding

    • The N-terminal region (Ile32-Ala80) is important for TMED10 homo-oligomerization

How can I use TMED10 antibodies to investigate its role in neurodegenerative diseases?

TMED10 has been implicated in Alzheimer's disease (AD) pathology. To investigate this connection:

• Expression analysis in disease models:

  • Western blot analysis shows decreased TMED10 expression in AD patient brain tissue

  • Immunohistochemistry can be used to visualize TMED10 distribution in brain sections

• Functional studies of TMED10 in Aβ production:

  • TMED10 depletion increases Aβ production, which can be measured by ELISA or immunoblotting

  • Treatment of cells with Aβ reduces the interaction between TMED10 and ATG4B

• Autophagy modulation:

  • TMED10 negatively regulates autophagy by inhibiting ATG4B activity

  • The interaction between TMED10 and ATG4B can be monitored using co-IP or BiFC assays

  • Autophagy markers (LC3-II/LC3-I ratio) can be assessed in relation to TMED10 levels

• Correlation studies in patient samples:

  • Compare TMED10 levels with disease markers in patient-derived samples

  • Analyze TMED10 polymorphisms in relation to disease risk or progression

• Therapeutic targeting approaches:

  • Use peptides derived from TMED10's extracellular domain to modulate its function

  • For example, peptides from amino acids 91-110 of TMED10 inhibit TGF-β-induced Smad2 phosphorylation

How do I interpret contradictory results between different TMED10 antibodies?

Contradictory results can arise from several factors:

• Epitope differences:

  • Antibodies targeting different regions of TMED10 may give varying results

  • Center region antibodies (e.g., against amino acids 101-200) versus C-terminal antibodies may detect different forms of the protein

  • Create a table mapping your antibodies to their specific epitopes:

• Post-translational modifications:

  • TMED10 undergoes various modifications that may mask certain epitopes

  • Different tissues/cell types may exhibit different modification patterns

• Protein complexes:

  • TMED10 forms complexes with other proteins, potentially obscuring epitopes

  • Denaturation conditions in western blotting versus native conditions in IP may yield different results

• Validation strategy:

  • When faced with contradictory results:

    • Use genetic models (knockdown/knockout) to determine specificity

    • Test antibodies under identical conditions

    • Consider using mass spectrometry to confirm protein identity

    • Assess if differences correlate with biological variables (e.g., disease state, activation condition)

What are the experimental considerations when using TMED10 antibodies in T cell function studies?

Recent research has revealed TMED10's role in regulating PD-1 expression in T cells . When investigating this aspect:

• T cell activation conditions:

  • TMED10 expression and localization may change upon T cell activation

  • Use standardized activation protocols (e.g., CD3/CD28 stimulation, PMA/ionomycin)

  • Monitor activation markers alongside TMED10 detection

• Cell surface versus total TMED10:

  • Use cell surface biotinylation to distinguish membrane-localized from intracellular TMED10

  • Flow cytometry analysis of intact versus permeabilized cells can differentiate surface from total protein

• Functional readouts:

  • Cytokine production (IFN-γ, TNF, IL-2) is enhanced in TMED10-deficient T cells

  • PD-1 surface expression is reduced in TMED10 knockout T cells

  • Correlate these functional changes with TMED10 levels and localization

• Tumor cell co-culture models:

  • Co-culture of T cells with tumor cells (e.g., B16F10-OVA) reveals functional consequences of TMED10 perturbation

  • TMED10-deficient T cells show enhanced anti-tumor activity that correlates with reduced PD-1 expression

• Translational correlation:

  • Single-cell RNA analysis shows a positive correlation between TMED10 expression in tumor-infiltrating CD8 T cells and T cell dysfunction signatures

  • This correlates with poor responses to immune checkpoint blockade therapy

Why do I observe multiple bands when using TMED10 antibodies in western blotting?

Multiple bands in TMED10 western blots may represent:

• Protein processing:

  • TMED10 may undergo proteolytic processing resulting in fragments of different sizes

  • For example, when studying IGF2 trafficking, both pro-IGF2 and mature IGF2 forms can be detected

• Post-translational modifications:

  • Glycosylation, phosphorylation, or other modifications can alter migration patterns

  • Deglycosylation experiments with enzymes like PNGase F can help identify glycosylated forms

• Protein complexes resistant to denaturation:

  • TMED10 forms homo-oligomers and heteromeric complexes with other TMED proteins

  • Ensure complete denaturation with sufficient SDS, heat, and reducing agents

• Cross-reactivity:

  • Some antibodies may detect other TMED family members due to sequence similarity

  • Validate with TMED10 knockout/knockdown controls to identify specific bands

• Resolution approaches:

  • Use gradient gels for better separation of closely migrating bands

  • Compare results with multiple antibodies targeting different epitopes

  • Include appropriate positive controls (overexpression) and negative controls (knockdown)

What are the optimal fixation and permeabilization conditions for detecting TMED10 in immunofluorescence?

For optimal TMED10 detection in immunofluorescence:

• Fixation options:

  • 4% paraformaldehyde (10-15 minutes at room temperature) preserves morphology while maintaining antigenicity

  • Methanol fixation (-20°C for 10 minutes) may better expose some epitopes but can disrupt membrane structures

  • Test both methods to determine optimal conditions for your specific antibody

• Permeabilization:

  • For PFA-fixed cells: 0.1-0.2% Triton X-100 (10 minutes) for complete permeabilization

  • For membrane proteins: gentler permeabilization with 0.1% saponin may preserve membrane localization

  • Some antibodies work better with specific permeabilization methods

• Antigen retrieval:

  • For tissue sections: citrate buffer (pH 6.0) heat-induced epitope retrieval may improve detection

  • For cells: brief treatment with dilute SDS (0.1% for 5 minutes) can help expose masked epitopes

• Blocking recommendations:

  • Use 5% normal serum from the same species as the secondary antibody

  • Add 1% BSA to reduce non-specific binding

  • Include 0.1% Tween-20 in blocking and antibody solutions to reduce background

• Controls:

  • Include TMED10 knockdown/knockout cells as negative controls

  • Co-stain with markers for ER (calnexin), Golgi (GM130), or other compartments to confirm localization

How can I optimize co-immunoprecipitation protocols for studying TMED10 interactions?

To optimize co-IP for TMED10 protein interactions:

• Lysis conditions:

  • For membrane proteins like TMED10, use non-denaturing detergents:

    • 1% NP-40 or Triton X-100 for standard interactions

    • Milder detergents (0.5% CHAPS, 0.5% digitonin) for preserving more sensitive complexes

  • Include protease and phosphatase inhibitors to prevent degradation

• Antibody selection:

  • Choose antibodies validated for IP applications

  • Avoid antibodies whose epitopes may be involved in protein interactions

  • Consider using tagged versions (V5, HA, Myc) of TMED10 with tag-specific antibodies

• IP procedures:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Use proper antibody:bead ratios (typically 2-5 μg antibody per 20-50 μl beads)

  • For weak interactions, consider crosslinking approaches (e.g., DSP, formaldehyde)

• Washing stringency:

  • For strong interactions: more stringent washes (higher salt, more detergent)

  • For weak interactions: gentler washes to preserve complexes

  • Always include a final wash with lower detergent concentration

• Successful examples:

  • TMED10-ATG4B interactions were successfully detected using anti-HA, anti-V5, anti-Myc, and anti-Strep tag antibodies in overexpression systems

  • Endogenous TMED10-ATG4B interactions were detected using anti-TMED10 antibodies for IP followed by anti-ATG4B for western blot

  • TMED10 interactions with TGF-β receptors were demonstrated using co-IP approaches

How is TMED10 research evolving in the context of immune checkpoint regulation?

Recent research has uncovered an unexpected role for TMED10 in immune regulation:

What is known about the regulatory mechanisms controlling TMED10 expression and function?

Understanding TMED10 regulation is still emerging:

• Expression regulation:

  • Downregulation of TMED10 is observed in Alzheimer's disease patients

  • The mechanisms controlling this downregulation remain largely unknown

  • Future research should investigate transcriptional and post-transcriptional regulatory mechanisms

• Functional regulation:

  • TMED10 function appears to be regulated through:

    • Protein-protein interactions with other TMED family members

    • Changes in subcellular localization

    • Post-translational modifications

• Interdependency with other TMED proteins:

  • Depletion of TMED10 reduces levels of other TMED family members (TMED2, TMED9)

  • This suggests coordinated regulation or stability mechanisms among TMED proteins

  • Understanding this interdependency may reveal master regulators of the TMED system

• Response to cellular stress:

  • Autophagy activation conditions (rapamycin treatment, serum deprivation) diminish TMED10-ATG4B interactions

  • Aβ treatment induces dissociation of TMED10 from ATG4B

  • These findings suggest TMED10 functions as a stress-responsive regulatory protein

• Future directions:

  • Identification of upstream regulators of TMED10 expression

  • Characterization of post-translational modifications affecting TMED10 function

  • Development of tools to specifically modulate TMED10 activity without affecting protein levels

How can I apply multi-omics approaches to study TMED10 function comprehensively?

Integrative approaches can provide deeper insights into TMED10 biology:

• Proteomics approaches:

  • Proximity labeling methods (BioID, APEX) to identify the TMED10 interactome

  • Quantitative proteomics comparing wild-type and TMED10-depleted cells to identify affected pathways

  • Secretome analysis to identify proteins whose secretion depends on TMED10

• Transcriptomics integration:

  • RNA-seq of TMED10 knockout versus wild-type cells reveals downstream transcriptional changes

  • Single-cell RNA-seq can identify cell populations affected by TMED10 perturbation

  • Integration with proteomics data can highlight post-transcriptional regulatory mechanisms

• Structural biology approaches:

  • Cryo-EM or X-ray crystallography of TMED10 alone or in complex with interaction partners

  • Structural information can guide the design of specific inhibitors or modulating peptides

• Systems biology integration:

  • Network analysis to position TMED10 within cellular pathways

  • Mathematical modeling of protein trafficking with and without TMED10 function

  • Identification of key nodes that could be targeted alongside TMED10 for synergistic effects

• Translational approaches:

  • Correlation of TMED10 levels with clinical outcomes in various diseases

  • Development of TMED10-based biomarkers for disease diagnosis or prognosis

  • Design of TMED10-targeting therapeutics based on integrated multi-omics data