TMEM2 Antibody, HRP conjugated

What is TMEM2 and what cellular functions does it regulate?

TMEM2 (Transmembrane Protein 2, also known as CEMIP2) is a single-pass type II transmembrane protein with a short cytoplasmic portion and an extended extracellular portion. Its protein structure includes several recognized extracellular domains: a G8 domain, a Pander-like Tmem2 (PLT) domain, parallel beta helix (PbH1) repeats, and a second Pander-like (PL) domain . TMEM2 plays critical roles in multiple cellular processes, particularly in regulating extracellular matrix composition through hyaluronic acid (HA) metabolism.

Functionally, TMEM2 has been demonstrated to act as a cell surface regulator of hyaluronic acid levels. In zebrafish models, Tmem2 restricts atrioventricular canal (AVC) differentiation by regulating HA degradation, which subsequently confines Wnt signaling distribution . The protein localizes to multiple cellular compartments including the cytosol, plasma membrane, nucleolus, and can be found in extracellular exosomes . Emerging evidence suggests TMEM2 has important functions in developmental biology, cancer biology, and neurobiology through its involvement in cell signaling and membrane transport processes .

How do HRP-conjugated TMEM2 antibodies work in experimental protocols?

HRP (horseradish peroxidase)-conjugated TMEM2 antibodies function through enzymatic signal amplification in immunodetection techniques. When these antibodies bind to their TMEM2 target, the conjugated HRP enzyme catalyzes a colorimetric or chemiluminescent reaction when exposed to appropriate substrates. This enzymatic reaction generates a detectable signal proportional to the amount of TMEM2 present in the sample.

In Western blot applications, HRP-conjugated antibodies allow for the sensitive detection of TMEM2 protein. The detection involves using enhanced chemiluminescence (ECL) reagents that react with the HRP enzyme to produce light, which can be captured on film or with digital imaging systems. For example, in studies examining regulation of HA metabolism, HRP-conjugated secondary antibodies were used to detect primary TMEM2 antibodies, allowing researchers to visualize changes in TMEM2 protein expression under different experimental conditions .

What are the recommended sample preparation protocols for optimal TMEM2 detection?

For optimal TMEM2 detection, proper sample preparation is crucial due to the protein's multiple cellular localizations and large molecular weight (~154 kDa calculated, observed at ~200 kDa) . The following protocol has been validated for TMEM2 detection in various applications:

Tissue/Cell Lysis Protocol:

• Harvest cells or tissue (mouse lung has been verified as a positive control sample)

• Lyse samples in a buffer containing adequate protease inhibitors

• Homogenize thoroughly to ensure complete protein extraction

• Centrifuge to remove cellular debris

• Quantify protein concentration using standard methods (Bradford or BCA assay)

Western Blot Sample Preparation:

• Dilute protein lysates to 20-50 μg per lane

• Mix with reducing sample buffer

• Heat at 95°C for 5 minutes

• Cool on ice before loading onto gels

For immunohistochemistry applications, formalin-fixed paraffin-embedded (FFPE) sections should undergo appropriate antigen retrieval (heat-induced epitope retrieval in citrate buffer pH 6.0) to expose the TMEM2 epitopes that may be masked during the fixation process .

What are the recommended dilutions and application conditions for TMEM2 antibodies?

Based on validated protocols, the following application-specific dilutions and conditions are recommended for TMEM2 antibodies:

The TMEM2 Rabbit Polyclonal Antibody has been validated for detecting mouse and rat TMEM2, with a synthetic peptide corresponding to a sequence within amino acids 1150-1250 of human TMEM2 (NP_037522.1) used as the immunogen . When using HRP-conjugated secondary antibodies to detect primary TMEM2 antibodies, a dilution range of 1:2000-1:5000 is typically effective for Western blot applications .

How can researchers differentiate between the enzymatic and regulatory functions of TMEM2 in hyaluronic acid metabolism?

Differentiating between TMEM2's potential enzymatic and regulatory functions requires carefully designed experimental approaches due to species-specific differences in TMEM2 function. Current research indicates that while mouse TMEM2 possesses direct HA-degrading activity, human TMEM2 appears to function primarily as a regulator rather than a catalytic enzyme .

Recommended Experimental Approach:

• Domain Swapping Analysis: Construct chimeric proteins containing different domains from mouse and human TMEM2 to identify functional regions. For example, research has shown that chimeras with mouse GG domains display HA-degrading activity, suggesting domain-specific functions .

• Point Mutation Studies: Generate specific mutations in key residues such as R267H in the PLT domain, which has been shown to affect hyaluronidase activity. In zebrafish models, this mutation impaired TMEM2's efficacy in restricting AVC differentiation while still supporting cardiac fusion, indicating context-dependent requirements for the hyaluronidase activity .

• Knockdown and Rescue Experiments: Use siRNA to knockdown endogenous TMEM2 and then perform rescue experiments with wild-type and mutated constructs. This approach revealed that TMEM2 knockdown in human dermal fibroblasts abrogated the effects of proinflammatory cytokines and TGF-β on HA metabolism .

• Extracellular HA Measurement: Quantify changes in extracellular HA levels in response to TMEM2 manipulation. Studies in TMEM2-knockdown NHDFs showed that changes in HAS2 mRNA levels correspond to alterations in extracellular HA amounts in culture medium .

When designing these experiments, researchers should consider that:

• Human and mouse TMEM2 share approximately 66% nucleotide homology and 87% amino acid homology

• Both contain similar domain structures but may have evolved divergent functions

• The GG domain appears particularly important for enzymatic activity in mouse TMEM2

What methodological approaches are most effective for studying TMEM2's role in developmental processes?

Studying TMEM2's developmental roles requires integrating multiple methodological approaches, particularly given its crucial function in processes such as atrioventricular canal differentiation. Based on successful research strategies, the following approaches are recommended:

1. In vivo Zebrafish Model System:
Zebrafish embryos provide an excellent model for studying TMEM2's developmental functions due to their external development and optical transparency. Key methodologies include:

• Microinjection of mRNA (150-300 pg) encoding full-length or variant TMEM2 at the one-cell stage

• For visualization of subcellular localization, lower doses (50 pg) of mRNA are recommended

• In situ hybridization to examine expression patterns of genes regulated by TMEM2, such as myl7 and bmp4

• Live imaging to track developmental processes in real-time

2. Structure-Function Analysis:
To determine which domains of TMEM2 mediate specific developmental functions:

• Generate truncated or chimeric TMEM2 constructs

• Perform site-directed mutagenesis of key residues (e.g., R267H in the PLT domain)

• Express these variants in TMEM2-deficient backgrounds and assess rescue efficiency

3. Hyaluronic Acid Manipulation:
To test the hypothesis that TMEM2 functions through HA regulation:

• Visualize HA localization using biotinylated HA binding protein (HABP)

• Perform gain-of-function experiments with exogenous hyaluronidase (e.g., injection of ~50 units of hyaluronidase from Streptomyces hyalurolyticus into the pericardial sac)

• Assess whether hyaluronidase treatment can rescue TMEM2 mutant phenotypes

4. Signaling Pathway Analysis:
To understand how TMEM2 interacts with developmental signaling pathways:

• Examine Wnt signaling distribution using reporter constructs

• Analyze BMP pathway activity through expression of downstream targets

• Investigate how TMEM2 manipulation affects these signaling pathways

This integrated approach has successfully revealed that TMEM2's ectodomain is critical for restricting AVC differentiation through regulation of HA levels, which subsequently influences Wnt and BMP signaling distribution .

How can discrepancies in observed molecular weights of TMEM2 be addressed in experimental design?

Discrepancies between the calculated and observed molecular weights of TMEM2 (calculated ~154 kDa vs. observed ~200 kDa) represent a common challenge in TMEM2 research that requires careful experimental design. These differences likely arise from post-translational modifications, particularly glycosylation of this transmembrane protein.

Recommended Protocol for Addressing Molecular Weight Discrepancies:

• Comprehensive Molecular Weight Analysis:

  • Run protein samples alongside precisely calibrated molecular weight markers

  • Use gradient gels (4-15%) to achieve better resolution of high molecular weight proteins

  • Include positive control samples (e.g., mouse lung tissue) with verified TMEM2 expression

• Glycosylation Analysis:

  • Treat protein lysates with glycosidases (PNGase F for N-linked glycans, O-glycosidase for O-linked glycans)

  • Compare migration patterns before and after deglycosylation

  • Use lectins to detect and characterize specific glycan structures on TMEM2

• Domain-Specific Detection:

  • Employ antibodies targeting different epitopes of TMEM2 to verify full-length protein detection

  • The synthetic peptide corresponding to amino acids 1150-1250 of human TMEM2 (ERVKIQAATDSKDISNCMAKAYPQYYRKPSVVKRMPAMLTGLCQGCGTRQVVFTSDPHKSYLPVQFQSPDKAETQRGDPSVISVNGTDFTFRSAGVLLLVV) represents one validated epitope region

• Alternative Detection Methods:

  • Complement Western blot with immunoprecipitation followed by mass spectrometry

  • Express tagged versions of TMEM2 (His, FLAG, or GFP) for orthogonal detection methods

  • Consider native gel electrophoresis to preserve protein complexes that might affect migration

• Data Interpretation Guidelines:

  • When reporting TMEM2 detection, always note both the calculated and observed molecular weights

  • Include representative Western blot images with molecular weight markers clearly indicated

  • Document all experimental conditions that might affect protein migration (reducing vs. non-reducing conditions, gel percentage, buffer systems)

By systematically addressing these potential sources of molecular weight variation, researchers can ensure more consistent and interpretable results when studying TMEM2 proteins across different experimental systems.

What controls and validation methods are essential when studying TMEM2's interaction with inflammatory pathways?

When investigating TMEM2's role in inflammatory responses, rigorous controls and validation methods are essential, particularly given evidence that TMEM2 mediates the effects of proinflammatory cytokines on hyaluronic acid metabolism .

Essential Controls and Validation Methods:

• Knockdown/Knockout Validation:

  • Confirm TMEM2 knockdown efficiency at both mRNA and protein levels

  • Use multiple siRNA sequences targeting different regions of TMEM2 to rule out off-target effects

  • Include non-targeting siRNA controls

  • Rescue experiments with siRNA-resistant TMEM2 constructs to confirm specificity

• Cytokine Stimulation Controls:

  • Use dose-response curves to determine optimal cytokine concentrations

  • Time-course experiments (6h, 12h, 24h) to capture temporal dynamics of responses

  • Include both individual cytokines (e.g., IL-1β alone) and cocktails (IL-1β, IL-6, TNF-α)

  • Validate cytokine activity using established readouts (e.g., NF-κB activation)

• Downstream Target Validation:

  • Monitor both mRNA and protein levels of key target genes (HAS2, HYBID)

  • Measure functional outcomes such as extracellular HA levels

  • Cross-validate findings using both RT-qPCR and Western blot analysis

• Cell Type Considerations:

  • Normal human dermal fibroblasts (NHDFs) have been validated for studying TMEM2's role in inflammatory responses

  • Compare responses across multiple cell types when possible

  • Consider primary cells versus cell lines

• Pathway Validation:

  Pathway Component	Validation Approach	Expected Outcome with TMEM2 Knockdown
  Inflammatory cytokines (IL-1β, IL-6, TNF-α)	RT-qPCR, ELISA	Abrogation of cytokine-induced gene expression changes
  TGF-β signaling	SMAD phosphorylation, target gene expression	Partial recovery of HYBID expression
  HAS2 activation	RT-qPCR, HA quantification	Modulation of HAS2 expression and HA production

• Technical Replicates and Statistical Analysis:

  • Minimum of three biological replicates

  • Appropriate statistical tests (typically ANOVA with post-hoc tests for multiple comparisons)

  • Clear reporting of p-values and variation measures

These comprehensive controls have successfully revealed that TMEM2 plays a regulatory role in human cells, where it mediates the effects of inflammatory cytokines on HYBID expression and HA production, rather than directly functioning as a catalytic hyaluronidase .