TMEM161B Antibody

What is TMEM161B and why is it significant for research?

TMEM161B (Transmembrane protein 161b) is a highly conserved protein with eight transmembrane domains that has emerged as an essential regulator in multiple biological systems. It has been identified in high-throughput phenotypic screens across fly, zebrafish, and mouse models. Its significance lies in its crucial role in regulating cardiac rhythm and morphology through modulation of intracellular calcium ion handling. Additionally, homozygous or heterozygous missense mutations in TMEM161B have been associated with structural brain malformations in humans, including polymicrogyria, intractable seizures, microcephaly, and hypotonia. Its diverse functions across species make it an important target for research in both cardiovascular and neurological contexts .

What types of TMEM161B antibodies are currently available for research?

Several types of TMEM161B antibodies are available, varying in host species, clonality, and target epitopes:

• Polyclonal antibodies raised in rabbits targeting various amino acid regions including AA 35-84, AA 71-120, and N-terminal regions

• Monoclonal antibodies (such as clone R08-6J4) for applications requiring higher specificity

• Antibodies with different conjugates including unconjugated, HRP-conjugated, FITC-conjugated, and biotin-conjugated versions for various detection methods

• Immunoaffinity purified antibodies that target specific epitopes, such as the synthetic peptide located between aa35-84 of human TMEM161B

What is the species cross-reactivity profile of TMEM161B antibodies?

TMEM161B antibodies demonstrate significant cross-reactivity across species due to the high conservation of the protein. Based on BLAST analysis, antibodies targeting regions like AA 35-84 show 100% identity with TMEM161B in human, chimpanzee, gorilla, gibbon, monkey, galago, marmoset, mouse, elephant, dog, bovine, bat, horse, and guinea pig. They also show high identity (92%) with rabbit and pig TMEM161B, and moderate identity (85%) with chicken TMEM161B. Some antibodies even demonstrate cross-reactivity with zebrafish (84%) and Xenopus (80%) . This cross-reactivity profile makes these antibodies valuable for comparative studies across multiple model organisms .

What are the optimal applications for TMEM161B antibodies in cardiac research?

For cardiac research investigating TMEM161B's role in rhythm regulation and morphology, the optimal approach involves a multi-method strategy. Western blotting using antibodies targeting the N-terminal or AA 35-84 regions can confirm TMEM161B expression levels in cardiac tissue at a recommended dilution of 0.2-1 μg/mL . Immunohistochemistry on paraffin-embedded cardiac sections helps visualize TMEM161B localization within cardiomyocytes, which is crucial for understanding its membrane localization. For functional studies, coupling antibody-based detection with calcium imaging using gCaMP reporters provides insights into how TMEM161B affects calcium transients in cardiomyocytes . This integrated approach allows researchers to correlate TMEM161B expression patterns with its effects on potassium and calcium ion currents, which are essential for cardiac action potential dynamics .

How should researchers validate the specificity of TMEM161B antibodies?

Validation of TMEM161B antibody specificity requires a comprehensive approach:

• Control tissues/cells: Compare staining patterns between tissues known to express TMEM161B (based on RNA data) and those with minimal expression.

• Knockout/knockdown validation: Ideally, use TMEM161B knockout or knockdown models as negative controls. The grime zebrafish model or mouse models with TMEM161B mutations can serve as valuable validation tools .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide prior to application to confirm that binding is specifically blocked.

• Multiple antibody comparison: Use antibodies targeting different epitopes of TMEM161B (such as those targeting AA 35-84 versus N-terminal regions) to confirm consistent staining patterns .

• Western blot verification: Confirm that the antibody detects a protein of the expected molecular weight (~53 kDa for human TMEM161B) and that this band disappears or is reduced in knockout/knockdown samples.

• Cross-species validation: Given the high conservation of TMEM161B across species, consistent detection across different species adds credibility to antibody specificity .

What are the recommended sample preparation protocols for immunohistochemistry with TMEM161B antibodies?

For optimal immunohistochemistry results with TMEM161B antibodies:

• Fixation: Use 4% paraformaldehyde for 24-48 hours at room temperature for tissue samples.

• Embedding and sectioning: Following standard paraffin embedding protocols, section tissues at 4-6 μm thickness.

• Antigen retrieval: Perform heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes at 95-100°C, which is essential as the eight transmembrane domains of TMEM161B can be masked during fixation.

• Blocking: Block with 5% normal serum (from the same species as the secondary antibody) in PBS with 0.1% Triton X-100 for 1 hour at room temperature.

• Primary antibody incubation: Dilute TMEM161B antibodies at 1:20-1:50 as recommended for immunohistochemistry applications , and incubate overnight at 4°C.

• Detection system: Use a sensitive detection system compatible with your primary antibody, such as HRP-conjugated secondary antibodies followed by DAB visualization.

• Controls: Always include negative controls (omitting primary antibody) and positive controls (tissues known to express TMEM161B) .

How can researchers distinguish between specific and non-specific binding when using TMEM161B antibodies?

Distinguishing between specific and non-specific binding requires systematic evaluation:

• Signal pattern assessment: Specific TMEM161B staining should be consistent with its known subcellular localization as a transmembrane protein. For TMEM161B, expect membrane-associated staining patterns rather than diffuse cytoplasmic or nuclear signals.

• Comparison with transcript data: The protein expression pattern should generally correlate with mRNA expression patterns from databases or RT-PCR results.

• Titration experiments: Perform antibody titration experiments to determine the optimal concentration that maximizes specific signal while minimizing background. The recommended range of 0.2-1 μg/mL for Western blotting serves as a starting point .

• Blocking peptide controls: Using the immunizing peptide (such as the synthetic peptide corresponding to amino acids 35-84) as a competitive inhibitor should abolish specific signals but not affect non-specific binding .

• Knockout/knockdown comparison: The most definitive validation comes from comparing staining between wild-type and TMEM161B-deficient samples. Signals that persist in knockout samples are non-specific.

• Cross-antibody validation: Confirm findings using multiple antibodies targeting different epitopes of TMEM161B to rule out epitope-specific artifacts.

What are common issues when working with TMEM161B antibodies and how can they be resolved?

Common issues and their solutions include:

• Weak or absent signal:

  • Increase antibody concentration (start with manufacturer's recommended dilution of 1:20-1:50 for IHC )

  • Optimize antigen retrieval methods (critical for membrane proteins like TMEM161B)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use more sensitive detection systems

• High background:

  • Increase blocking time and concentration (5% skim milk for Western blots )

  • Reduce primary antibody concentration

  • Add 0.1-0.3% Triton X-100 to reduce non-specific binding

  • Increase wash steps duration and number

• Multiple bands on Western blot:

  • Use freshly prepared samples with protease inhibitors

  • Check for post-translational modifications of TMEM161B

  • Optimize SDS-PAGE conditions for transmembrane proteins

  • Use gradient gels to better separate proteins

• Variability between experiments:

  • Standardize tissue collection and fixation protocols

  • Prepare larger batches of antibody dilutions

  • Include positive controls in each experiment

  • Maintain consistent incubation times and temperatures

• Cross-reactivity issues:

  • Use antibodies with validated specificity for your species of interest

  • Perform pre-adsorption controls

  • Consider using more specific monoclonal antibodies

How should researchers interpret TMEM161B expression patterns in relation to calcium handling phenotypes?

Interpretation of TMEM161B expression in relation to calcium handling requires careful consideration:

How can TMEM161B antibodies be utilized to investigate the molecular pathways linking cardiac rhythm and brain development?

TMEM161B antibodies offer unique opportunities to investigate the shared molecular pathways between cardiac rhythm regulation and brain development:

• Comparative tissue analysis: Using immunohistochemistry with TMEM161B antibodies to simultaneously examine expression patterns in cardiac and neural tissues across developmental stages can reveal tissue-specific or shared expression domains. This approach can identify common signaling hubs where TMEM161B functions in both systems .

• Co-immunoprecipitation studies: TMEM161B antibodies can be used in co-IP experiments to identify protein interaction partners in heart versus brain tissue, potentially revealing shared or tissue-specific molecular complexes. This is particularly relevant given TMEM161B's role in calcium handling, which is crucial for both cardiac function and neuronal signaling .

• Patient-derived cell models: In cells derived from patients with TMEM161B mutations (associated with brain malformations), antibodies can help track mislocalization or abnormal expression patterns of the mutant protein, potentially explaining how the same mutations can affect both cardiac and neural function .

• Calcium imaging correlation: Combining TMEM161B immunolabeling with calcium imaging in both cardiomyocytes and neurons can establish whether calcium handling defects are consistent across tissues, providing a mechanistic link between cardiac arrhythmias and neurological phenotypes seen in patients .

• Developmental timing studies: Using TMEM161B antibodies to track protein expression throughout development can reveal critical windows when TMEM161B function is essential in both cardiac and neural tissues, potentially explaining the temporal aspects of disease manifestation.

What approaches can researchers use to investigate the relationship between TMEM161B and its antisense RNA TMEM161B-AS1?

Investigating the relationship between TMEM161B and its antisense RNA TMEM161B-AS1 requires sophisticated approaches:

• Dual immunofluorescence and RNA-FISH: Combine TMEM161B antibody labeling with fluorescent in situ hybridization for TMEM161B-AS1 to determine spatial correlation between protein and antisense RNA expression. This approach can reveal whether they are co-expressed in the same cells or tissues .

• Expression correlation analysis: Quantify TMEM161B protein levels (using validated antibodies in Western blotting) and TMEM161B-AS1 RNA levels (using qRT-PCR) across multiple tissue samples to determine if there is a positive, negative, or neutral correlation between their expression levels.

• Perturbation studies: Artificially modulate TMEM161B-AS1 expression (overexpression or knockdown) and monitor changes in TMEM161B protein levels using antibody-based methods. This can establish whether the antisense RNA regulates TMEM161B translation or stability.

• Chromatin immunoprecipitation (ChIP): Use antibodies against chromatin modifiers alongside TMEM161B-AS1 detection to investigate whether the antisense RNA influences TMEM161B expression through epigenetic mechanisms.

• RNA-protein interaction studies: Investigate whether TMEM161B-AS1 directly interacts with TMEM161B protein or affects its interaction with other proteins using RNA immunoprecipitation with TMEM161B antibodies.

• Cancer context analysis: Given that TMEM161B-AS1 has been implicated in cancer progression, compare TMEM161B protein expression in cancer cells with differential TMEM161B-AS1 expression to understand potential disease implications .

How can researchers investigate the potential role of TMEM161B in protecting against oxidative stress?

To investigate TMEM161B's potential role in oxidative stress protection, researchers can employ these methodological approaches:

• Oxidative challenge experiments: Compare the response to oxidative stressors (e.g., H₂O₂, paraquat) between wild-type cells/tissues and those with TMEM161B knockdown/knockout, using TMEM161B antibodies to confirm protein depletion. This builds on observations that the related protein TMEM161A increases upon oxidative stress .

• Expression correlation: Use TMEM161B antibodies in immunoblotting or immunohistochemistry to quantify whether TMEM161B protein levels change in response to oxidative challenges, similar to the reported upregulation of TMEM161A under oxidative stress conditions .

• Calcium and ROS co-imaging: Since TMEM161B regulates calcium handling , and calcium dysregulation can promote oxidative stress, researchers can use TMEM161B antibodies to correlate protein expression with simultaneous calcium and reactive oxygen species (ROS) measurements in live cells.

• Subcellular localization changes: Examine whether oxidative stress induces changes in TMEM161B subcellular localization using immunofluorescence with compartment-specific markers (mitochondria, ER, plasma membrane).

• Post-translational modification analysis: Investigate whether oxidative stress induces post-translational modifications of TMEM161B (phosphorylation, ubiquitination) using immunoprecipitation with TMEM161B antibodies followed by mass spectrometry.

• Comparative analysis with TMEM161A: Use antibodies against both TMEM161B and TMEM161A to determine if they have complementary or redundant roles in oxidative stress response, given their 48% amino acid similarity and TMEM161A's known response to oxidative stress .

What techniques can researchers employ to determine the precise mechanism by which TMEM161B regulates calcium and potassium ion currents?

To elucidate the mechanism of TMEM161B regulation of ion currents, researchers can employ these advanced techniques:

• Proximity labeling proteomics: Use TMEM161B antibodies to validate BioID or APEX2 proximity labeling experiments that can identify proteins physically interacting with TMEM161B at the membrane, potentially revealing direct interactions with ion channels or regulatory proteins.

• Super-resolution microscopy: Combine TMEM161B antibody labeling with super-resolution imaging techniques (STORM, PALM) to visualize the nanoscale organization of TMEM161B relative to calcium channels (L-type calcium channels) and potassium channels (hERG/KCNH2) implicated in the IKr currents that TMEM161B regulates .

• Patch-clamp electrophysiology with acute antibody treatment: Apply function-blocking TMEM161B antibodies during patch-clamp recordings to determine if acute interference with surface-exposed domains of TMEM161B alters calcium or potassium currents.

• Domain mapping studies: Use antibodies targeting different domains of TMEM161B to correlate the expression/localization of specific protein regions with functional outcomes in calcium and potassium handling through imaging and electrophysiology.

• Mutagenesis validation: Generate cells expressing TMEM161B with mutations in key transmembrane domains and use antibodies to confirm expression and localization before assessing changes in ion channel function using electrophysiological methods.

• Heterologous expression systems: Co-express TMEM161B with specific calcium or potassium channel subunits in expression systems, then use antibodies to confirm successful expression before measuring channel function with electrophysiology to determine direct regulatory effects.