tmem182 Antibody

What is TMEM182 and where is it primarily expressed?

TMEM182 (Transmembrane Protein 182) is a 229 amino acid protein with a molecular weight of approximately 25.9 kDa, localized in the cell membrane . It is specifically expressed in skeletal muscle and adipose tissue and is regulated at the transcriptional level by the myogenic regulatory factor MyoD1 . TMEM182 has multiple isoforms with observed molecular weights of approximately 15 kDa and 26 kDa . The protein has been implicated in muscle organ development, cell differentiation regulation, and adipose tissue metabolism .

What are the known biological functions of TMEM182?

TMEM182 functions as a negative regulator in several biological processes:

• Muscle development: TMEM182 inhibits myoblast differentiation and fusion, induces muscle fiber atrophy, and delays muscle regeneration .

• Cardiac differentiation: TMEM182 disrupts the balance of Wnt/β-catenin signaling during myocardial differentiation of human iPS cells, inhibiting differentiation into cardiac progenitor cells and cardiomyocytes .

• Adipose metabolism: TMEM182 promotes fat deposition and plays a crucial role in regulating fat metabolism .

Mechanistically, TMEM182 directly interacts with integrin beta 1 (ITGB1), modulating its activation by coordinating the association between ITGB1 and laminin and regulating intracellular signaling .

What post-translational modifications occur in TMEM182?

TMEM182 undergoes post-translational modifications, primarily glycosylation, which may affect its function and subcellular localization . This modification is particularly relevant when considering antibody selection for detection, as glycosylation patterns may affect epitope accessibility.

What types of TMEM182 antibodies are available for research?

There are several types of TMEM182 antibodies available for research applications:

When selecting an antibody, researchers should consider the specific application, target region, species reactivity, and clonality based on experimental needs .

How should I validate a TMEM182 antibody for my specific application?

Validation of TMEM182 antibodies should follow these methodological steps:

• Positive control selection: Use tissues known to express TMEM182 (skeletal muscle, adipose tissue) as positive controls .

• Knockout/knockdown validation: Compare antibody reactivity in wild-type vs. TMEM182-knockout or knockdown samples to confirm specificity .

• Cross-reactivity testing: Test across multiple species if working with non-human models .

• Multiple detection methods: Validate using more than one technique (e.g., WB and IHC) .

• Optimization experiments: Determine optimal working dilutions experimentally for each application (typical ranges: WB: 1:200-1:1000, IHC: 1:20-1:200) .

Proper validation ensures experimental reliability and reproducibility when working with TMEM182 antibodies.

What are the optimal protocols for Western blot detection of TMEM182?

For optimal Western blot detection of TMEM182:

• Sample preparation: Use RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, protease inhibitor cocktail, 1mM PMSF, and 1mM Na₃VO₄) for protein extraction .

• Loading control: Include appropriate loading controls (GAPDH, α-tubulin, or Na,K-ATPase for membrane fractions) .

• Expected bands: Look for bands at approximately 15 kDa and 26 kDa, corresponding to different isoforms of TMEM182 .

• Antibody dilution: Start with dilutions of 1:200-1:1000 for primary antibody and optimize as needed .

• Incubation conditions: Probe overnight at 4°C with primary antibody for best results .

Note that sample-dependent optimization may be necessary, and researchers should consider running pilot experiments to determine optimal conditions for their specific samples .

What considerations are important for immunohistochemical detection of TMEM182?

For successful immunohistochemical detection of TMEM182:

• Antigen retrieval: Use TE buffer at pH 9.0 for optimal results (alternatively, citrate buffer at pH 6.0 can be used) .

• Antibody dilution: Begin with dilutions between 1:20-1:200 and optimize based on signal-to-noise ratio .

• Positive control tissues: Include skeletal muscle or adipose tissue sections as positive controls .

• Counterstaining: DAPI or Hoechst can be used for nuclear counterstaining .

• Visualization: Use a confocal microscope for higher resolution images of membrane localization .

For staining live cells with anti-TMEM182, wash cells with PBS, incubate in blocking buffer (3% bovine serum albumin/PBS) for 15 minutes, perform antibody incubation on ice, fix with 4% PFA/PBS, and then incubate with secondary antibody .

How can TMEM182 antibodies be used to study muscle development and regeneration?

TMEM182 antibodies can be utilized to investigate muscle development and regeneration through several methodological approaches:

• Temporal expression analysis: Track TMEM182 expression during different stages of myoblast differentiation using Western blot or immunofluorescence .

• Co-localization studies: Perform dual immunostaining with TMEM182 antibodies and markers of muscle differentiation (e.g., MyHC) to analyze spatial relationships .

• Regeneration models: In muscle injury models, use TMEM182 antibodies to monitor expression changes during the regeneration process .

• Quantitative analysis: Measure cross-sectional area (CSA) of muscle fibers in TMEM182-overexpressing or knockout models to assess the impact on muscle fiber size .

Studies have shown that TMEM182 knockout in mice leads to significant increases in body weight, muscle mass, muscle fiber number, and muscle fiber diameter, while overexpression induces muscle fiber atrophy .

What is the relationship between TMEM182 and integrin beta 1, and how can this be studied?

The interaction between TMEM182 and integrin beta 1 (ITGB1) is a critical aspect of TMEM182's function in muscle development. To study this relationship:

• Co-immunoprecipitation: Use TMEM182 antibodies to pull down protein complexes and detect ITGB1 in the precipitate (or vice versa) .

• Proximity ligation assay: Detect in situ protein-protein interactions between TMEM182 and ITGB1.

• Domain mapping: The interaction requires an extracellular hybrid domain of ITGB1 (aa 387–470) and a conserved region (aa 52–62) within the large extracellular loop of TMEM182 .

• Functional analysis: Assess downstream signaling effects on FAK-ERK and FAK-Akt signaling axes during myogenesis .

Mechanistically, TMEM182 modulates ITGB1 activation by coordinating the association between ITGB1 and laminin. Deletion of TMEM182 increases the binding activity of ITGB1 to laminin and induces activation of key signaling pathways .

How does TMEM182 regulate cardiac differentiation, and how can antibodies help study this process?

TMEM182 inhibits myocardial differentiation of human iPSCs through Wnt/β-catenin signaling modulation. To investigate this process using TMEM182 antibodies:

• Expression timing: Monitor TMEM182 expression at different stages of cardiac differentiation (Days 2, 6, 12) using Western blot or immunofluorescence .

• Signaling pathway analysis: Assess the impact of TMEM182 on Wnt/β-catenin signaling by examining phosphorylation states of GSK-3β (Ser9) and β-catenin (Ser552) .

• Cardiac marker co-staining: Combine TMEM182 antibodies with cardiac markers (TNNT2, α-actinin) to evaluate differentiation progression .

• Integrin-linked kinase (ILK) analysis: Investigate the relationship between TMEM182 and ILK expression, as TMEM182 increases ILK expression, maintaining active Wnt/β-catenin signaling .

Experimental evidence shows that TMEM182 overexpression decreases expression of cardiomyocyte markers and disrupts cardiac sarcomere structure formation .

What are common issues when working with TMEM182 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with TMEM182 antibodies:

• Multiple bands in Western blot: TMEM182 has multiple isoforms with observed molecular weights of 15 kDa and 26 kDa. Validate band specificity using knockout/knockdown controls .

• Weak signal in immunostaining:

  • Optimize antigen retrieval (TE buffer pH 9.0 recommended)

  • Increase antibody concentration (within 1:20-1:200 range for IHC)

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

• Background issues:

  • Use 3% bovine serum albumin/PBS for blocking (15 min)

  • Optimize secondary antibody dilution

  • Include additional washing steps

• Cross-reactivity: Validate antibody specificity using TMEM182-knockout tissues or cells as negative controls .

• Freeze-thaw degradation: Avoid repeated freeze-thaw cycles by aliquoting antibodies before storage .

What are the optimal storage and handling conditions for TMEM182 antibodies?

For maximum stability and performance of TMEM182 antibodies:

• Storage temperature: Store antibodies at -20°C for long-term storage .

• Short-term storage: For up to one week, antibodies can be stored at 2-8°C .

• Aliquoting: After receiving, aliquot antibodies to avoid repeated freeze-thaw cycles .

• Buffer conditions: Most commercial TMEM182 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Working solution: Dilute antibodies according to application requirements immediately before use .

• Shelf life: Typical validity is 12 months from the date of receipt when stored properly .

Note that sodium azide in antibody buffers is a POISONOUS AND HAZARDOUS SUBSTANCE and should be handled by trained staff only .

How can TMEM182 antibodies be used to study metabolic disorders and obesity?

TMEM182 plays a role in fat metabolism and deposition, making it relevant for metabolic research:

• Adipocyte differentiation studies: Use TMEM182 antibodies to track expression changes during preadipocyte to adipocyte conversion .

• Fat deposition analysis: Compare TMEM182 expression in normal versus obese tissue samples using immunohistochemistry .

• Pathway analysis: Investigate the relationship between TMEM182 and ECM-receptor interaction and cell adhesion pathways in adipocytes .

• Metabolomic correlation: Correlate TMEM182 expression with changes in metabolite profiles, particularly amino acids and lipid compositions .

Recent studies have shown that TMEM182 overexpression increases expression of fat synthesis-related genes and promotes differentiation of preadipocytes into fat cells, while TMEM182 knockout mice show significant decreases in abdominal fat deposition .

What competing or contradictory findings exist regarding TMEM182 function and how might antibody-based experiments resolve these?

There are several areas where additional antibody-based research could help resolve research questions about TMEM182:

• Tissue specificity: While TMEM182 is reported to be specifically expressed in muscle and adipose tissue , some data shows expression in liver cancer tissue . Immunohistochemical profiling across a broader tissue panel could resolve this apparent contradiction.

• Developmental timing: TMEM182 shows inhibitory effects when overexpressed from early stages of cardiac differentiation, but not when introduced at middle stages . Time-course immunostaining could better characterize the temporal specificity of TMEM182 function.

• Isoform-specific functions: The presence of multiple isoforms (15 kDa and 26 kDa) raises questions about potential functional differences. Isoform-specific antibodies could help distinguish their respective roles.

• Species differences: While TMEM182 gene orthologs have been reported in multiple species , functional conservation across species requires further investigation. Cross-species immunostaining could help establish evolutionary conservation of expression patterns.

Resolving these questions through careful antibody-based experiments would advance our understanding of TMEM182 biology and its potential as a therapeutic target for obesity-related diseases and muscle disorders.