TMEM64 Antibody

What is TMEM64 and what are its alternative designations?

TMEM64 stands for Transmembrane Protein 64, a protein that has several alternative designations in research literature. These include 9630015D15Rik, AI790744, AV300874, and DKFZp762C1112 . At the protein level, it has also been designated as CG11367 gene product . Understanding these alternative names is essential for comprehensive literature searches and avoiding overlooking relevant research when investigating this protein.

What cellular functions has TMEM64 been associated with?

TMEM64 has been primarily identified as a regulator for RANKL-mediated calcium signaling pathways. Research has demonstrated that TMEM64 directly associates with SERCA2 (Sarco/Endoplasmic Reticulum Ca²⁺-ATPase 2), which is critical for calcium homeostasis . Through this interaction, TMEM64 modulates intracellular calcium oscillations that are essential for osteoclast differentiation. Additionally, TMEM64 has been shown to affect mitochondrial reactive oxygen species (ROS) production following RANKL stimulation, suggesting its involvement in multiple cellular signaling cascades .

What experimental models are available for studying TMEM64 function?

Several experimental models have been developed for studying TMEM64 function:

• Knockout mouse models: Tmem64⁻/⁻ mice have been generated using embryonic stem cells with disruption of the Tmem64 gene. These models have been valuable in understanding the in vivo function of TMEM64, particularly in bone homeostasis .

• siRNA knockdown systems: Studies have utilized Tmem64 siRNA to investigate the effects of reduced Tmem64 expression on osteoclast generation and differentiation .

• Retroviral expression systems: Restoration of Tmem64 expression using MSCV-Tmem64 retroviral vectors in Tmem64⁻/⁻ cells has been employed to verify the specific role of Tmem64 in calcium signaling and osteoclast differentiation .

What specific antibodies are available for TMEM64 detection?

Several antibodies are available for TMEM64 detection with varying specificities and applications:

• Human-reactive TMEM64 antibodies applicable for ELISA, IHC, and Western blotting (e.g., ABIN7173257)

• Human-reactive TMEM64 antibodies specifically validated for ELISA applications (e.g., ABIN7173260)

• Specialized ELISA kits employing a two-site sandwich ELISA to quantitate TMEM64 in various sample types including cell culture supernatants, plasma, and serum

These antibodies have been validated through various methods to ensure specificity for TMEM64 detection with minimal cross-reactivity to analogous proteins .

How can I optimize TMEM64 antibody selection for specific experimental applications?

Selecting the appropriate TMEM64 antibody requires consideration of several factors:

• Species reactivity: Ensure the antibody has been validated for your species of interest. Available antibodies have specific reactivity profiles, with some targeting human TMEM64 and others targeting mouse Tmem64 .

• Application compatibility: Match the antibody to your intended application. Some antibodies are validated for multiple applications (ELISA, IHC, WB), while others are application-specific .

• Validation status: Prioritize antibodies with extensive validation data. For instance, antibodies with multiple validation studies (indicated by validation numbers in product listings) may provide more reliable results .

• Epitope location: Consider whether the target epitope is accessible in your experimental conditions, especially when dealing with transmembrane proteins that may have conformational constraints.

• Detection method compatibility: Ensure the antibody is compatible with your detection system (e.g., fluorescent vs. colorimetric detection) .

What are the optimal methods for measuring TMEM64 expression in different cell types?

Measuring TMEM64 expression requires technique selection based on the experimental question:

• For protein quantification:

  • Sandwich ELISA is optimal for quantitative measurement of TMEM64 in liquid samples including cell culture supernatants, plasma, and serum .

  • Western blotting can provide semi-quantitative data on TMEM64 protein levels and evaluate antibody specificity.

  • Immunohistochemistry allows visualization of TMEM64 localization within tissues and cells .

• For gene expression:

  • Real-time PCR and RT-PCR have been successfully employed to measure Tmem64 mRNA levels during osteoclast differentiation .

  • Northern blot analysis has been used to confirm Tmem64 knockout in experimental models .

• For dynamic studies:

  • Time-course experiments monitoring Tmem64 expression during differentiation processes have revealed that Tmem64 mRNA levels increase during osteoclast differentiation but decrease during osteoblast differentiation .

How can TMEM64 function be assessed in calcium signaling pathways?

Assessing TMEM64's role in calcium signaling requires specialized techniques:

• Calcium oscillation measurements: RANKL-induced [Ca²⁺]ᵢ oscillation can be measured in wild-type versus Tmem64⁻/⁻ cells to determine TMEM64's effect on calcium dynamics .

• Protein interaction studies: Co-immunoprecipitation or other protein-protein interaction assays can evaluate TMEM64's direct association with SERCA2 and other calcium-regulating proteins .

• Downstream signaling analysis: Western blotting for phosphorylated CaMKIV (calcium/calmodulin-dependent protein kinase IV) can assess the impact of TMEM64 on calcium-dependent signaling pathways .

• Mitochondrial ROS detection: Using mitochondrial ROS-specific dyes (such as MitoSOX) can help determine how TMEM64 affects RANKL-induced mitochondrial ROS production, which is linked to calcium signaling .

• Rescue experiments: Reintroducing TMEM64 expression in knockout cells should restore calcium oscillation and downstream effects if the phenotype is specifically due to TMEM64 deficiency .

How is TMEM64 involved in bone remodeling and osteoclast differentiation?

TMEM64 plays a critical role in bone remodeling through several mechanisms:

• Regulation of osteoclast differentiation: Tmem64⁻/⁻ mice exhibit reduced numbers of TRAP+ multinucleated cells (MNCs), indicating impaired osteoclast formation. This observation is supported by reduced serum TRACP-5b levels, an early marker of osteoclast formation .

• Modulation of calcium signaling: TMEM64 regulates RANKL-induced calcium oscillations that are essential for proper osteoclast differentiation. In Tmem64⁻/⁻ BMMs (bone marrow macrophages), calcium oscillation is impaired following RANKL stimulation .

• Activation of CaMKIV: TMEM64 is required for the activation of CaMKIV in response to RANKL stimulation. CaMKIV activation contributes to CREB activation, which is important for osteoclast differentiation .

• Mitochondrial ROS production: TMEM64 affects the production of mitochondrial reactive oxygen species following RANKL stimulation, which is linked to PGC1β upregulation and subsequent osteoclast differentiation .

• Impact on osteoblast function: Interestingly, Tmem64⁻/⁻ mice show increased osteoblast activity, as evidenced by increased osteoblast surface area, bone formation rates, and serum osteocalcin levels .

What are the challenges in detecting and quantifying TMEM64 in biological samples?

Researchers face several challenges when detecting and quantifying TMEM64:

• Protein conformation: As a transmembrane protein, TMEM64 may adopt different conformations depending on the cellular context or sample preparation, potentially affecting antibody recognition.

• Sample preparation optimization: Different biological samples (cell culture supernatants, plasma, serum) may require specific preparation protocols to effectively extract and preserve TMEM64 for analysis .

• Specificity concerns: Ensuring antibody specificity is crucial, as cross-reactivity with other TMEM family members (e.g., TMEM63A/B/C, TMEM62, etc.) could lead to false-positive results .

• Sensitivity limitations: Detection methods must be optimized for sensitivity, particularly when TMEM64 is expressed at low levels. Sandwich ELISA methods with amplification steps may be required for reliable quantification .

• Validation across experimental systems: Consistent validation across different experimental systems (in vitro cell cultures, animal models, human samples) is necessary to establish reliable detection protocols.

How do TMEM64 expression patterns differ across cell types and differentiation stages?

TMEM64 expression exhibits distinct patterns across different cell types and differentiation stages:

• Osteoclast lineage: Tmem64 mRNA levels increase during RANKL-induced osteoclast differentiation of bone marrow macrophages, suggesting its importance in osteoclast development .

• Osteoblast lineage: Conversely, Tmem64 expression is downregulated during osteoblast differentiation, indicating potentially divergent roles in different bone cell lineages .

• Temporal expression dynamics: The temporal expression pattern of TMEM64 during differentiation processes suggests its involvement in specific stages of cellular development and function.

• Tissue-specific expression: While the search results primarily focus on bone-related cells, the availability of antibodies reactive to human TMEM64 suggests its expression in human tissues that may be relevant for broader research applications .

What controls should be included when using TMEM64 antibodies in experimental studies?

Rigorous control strategies are essential when working with TMEM64 antibodies:

• Positive controls:

  • Cell lines or tissues with known TMEM64 expression

  • Recombinant TMEM64 proteins (available from sources like wheat germ or HEK-293 cells)

  • Samples from wild-type animals when comparing with knockout models

• Negative controls:

  • Samples from Tmem64 knockout animals or cells

  • Cells treated with Tmem64 siRNA to reduce expression

  • Isotype control antibodies to assess non-specific binding

• Validation controls:

  • Multiple antibodies targeting different epitopes of TMEM64 to confirm specificity

  • Antibody pre-absorption with recombinant TMEM64 to confirm specificity

  • Western blot analysis to confirm antibody specificity by molecular weight

• Technical controls:

  • For ELISA: standard curves using recombinant proteins, blank wells, and dilution linearity tests

  • For IHC/IF: secondary antibody-only controls to assess background

How can I design experiments to investigate TMEM64's interaction with SERCA2 and its impact on calcium signaling?

To investigate TMEM64-SERCA2 interactions and their functional consequences:

• Co-immunoprecipitation experiments:

  • Precipitate TMEM64 and probe for SERCA2 (and vice versa)

  • Include appropriate negative controls (IgG, unrelated proteins)

  • Consider crosslinking approaches for transient interactions

  • Analyze samples from different cellular contexts to assess context-specificity

• Proximity ligation assays:

  • Visualize and quantify TMEM64-SERCA2 interactions in situ

  • Compare interaction patterns across cell types and differentiation states

• Functional calcium assays:

  • Measure calcium fluxes in wild-type vs. TMEM64-deficient cells

  • Design rescue experiments with wild-type and mutant TMEM64 to identify critical interaction domains

  • Monitor SERCA2 activity in the presence and absence of TMEM64

• Structure-function studies:

  • Generate truncation or point mutations in TMEM64 to map interaction domains

  • Assess the impact of these mutations on SERCA2 binding and calcium signaling

• Pharmacological approaches:

  • Use SERCA inhibitors (e.g., thapsigargin) to determine if TMEM64 effects are dependent on SERCA2 function

  • Compare calcium dynamics in the presence/absence of TMEM64 under various pharmacological conditions

What are potential pitfalls in interpreting TMEM64 knockout/knockdown phenotypes?

Interpreting TMEM64 knockout or knockdown phenotypes requires careful consideration of:

• Compensatory mechanisms:

  • Other TMEM family members might compensate for TMEM64 loss

  • Alternative calcium signaling pathways may be upregulated

  • Analyze expression of related genes in TMEM64-deficient models

• Cell-autonomous vs. non-cell-autonomous effects:

  • Tmem64⁻/⁻ mice show both osteoclast defects and enhanced osteoblast activity

  • Co-culture experiments with wild-type and knockout cells can help distinguish intrinsic vs. extrinsic effects

  • Cell type-specific conditional knockout models may provide more precise insights

• Developmental vs. acute effects:

  • Germline knockout phenotypes may reflect developmental adaptations

  • Inducible knockout or acute knockdown approaches may reveal different phenotypes

  • Time-course analyses can help distinguish immediate vs. long-term consequences of TMEM64 loss

• Partial vs. complete loss-of-function:

  • siRNA typically achieves partial knockdown, while genetic knockout creates complete loss

  • Different phenotypic severity may reflect residual TMEM64 activity rather than different functions

  • Quantify the degree of TMEM64 reduction and correlate with phenotypic severity

• Experimental context dependencies:

  • Phenotypes may vary depending on cell type, differentiation stage, or culture conditions

  • RANKL concentration and timing can influence the observed effects on osteoclast differentiation

  • Standardize experimental conditions when comparing across studies

How might TMEM64 function be targeted for therapeutic development in bone disorders?

Considering TMEM64's role in bone remodeling, potential therapeutic strategies include:

• Modulating TMEM64-SERCA2 interaction:

  • Small molecules or peptides that enhance or inhibit this interaction could influence osteoclast differentiation

  • Specificity would be crucial to avoid disrupting calcium homeostasis in other tissues

• Targeting TMEM64 expression levels:

  • RNA-based therapeutics to modulate TMEM64 expression specifically in osteoclast precursors

  • Transcriptional regulators of TMEM64 could provide alternative targets

• Exploiting downstream pathways:

  • CaMKIV or CREB-targeted approaches might achieve similar effects without directly targeting TMEM64

  • Mitochondrial ROS modulation could influence TMEM64-dependent signaling pathways

• Combining with existing bone therapeutics:

  • TMEM64-targeted approaches might synergize with bisphosphonates or RANKL inhibitors

  • Dual targeting of osteoclast inhibition and osteoblast stimulation based on TMEM64's differential effects

• Biomarker development:

  • TMEM64 levels or activation status might serve as biomarkers for bone remodeling dynamics

  • ELISA-based detection methods could be adapted for clinical biomarker applications

What are unexplored areas of TMEM64 biology beyond bone homeostasis?

Beyond its established role in bone biology, several aspects of TMEM64 function remain unexplored:

• Expression and function in non-skeletal tissues:

  • Analyze TMEM64 expression across different human and animal tissues

  • Investigate potential roles in other calcium-dependent processes like muscle contraction, neuronal signaling, or immune cell function

• Involvement in other calcium-dependent pathologies:

  • Examine TMEM64 expression and function in calcium-related disorders such as cardiovascular disease, neurodegeneration, or cancer

  • Study TMEM64 polymorphisms in human populations for disease associations

• Regulation of TMEM64 expression and activity:

  • Identify transcriptional and post-transcriptional regulators of TMEM64

  • Investigate post-translational modifications that might regulate TMEM64 function

• Evolutionary conservation and divergence:

  • Compare TMEM64 structure and function across species to identify conserved domains and species-specific adaptations

  • Examine potential functional divergence among TMEM family members

• Subcellular dynamics and trafficking:

  • Study TMEM64 subcellular localization, trafficking, and turnover

  • Investigate how these processes might be regulated during differentiation or cellular stress