TMEM143 Antibody

What is TMEM143 and why is it important in research?

TMEM143 (Transmembrane protein 143) is a mitochondrially-located transmembrane protein belonging to the TMEM protein family . This protein family is significant because its members play important roles in the development and metastasis of cancer, particularly in transmitting signals between the extracellular environment and cytoplasmic proteins . Research into TMEM143 is valuable for understanding mitochondrial biology and potential roles in disease pathways, especially in cancer research contexts where transmembrane signaling is critical for cellular transformation and metastasis.

What applications can TMEM143 antibodies be used for?

TMEM143 antibodies have been validated for multiple experimental applications:

For optimal results, experimental conditions should be titrated for each specific application and sample type. TMEM143 antibodies have been successfully used to detect the protein in various human cell lines including HH, HepG2, Jurkat, and Ramos cells .

What is the expected molecular weight of TMEM143 in Western blot applications?

The calculated molecular weight of TMEM143 is 52 kDa, but the observed molecular weight typically ranges between 45-52 kDa in Western blot applications . This slight discrepancy between calculated and observed weights is common for transmembrane proteins due to post-translational modifications or the hydrophobic nature of membrane proteins affecting mobility during SDS-PAGE. When validating a new TMEM143 antibody, researchers should confirm bands within this range and consider using positive control samples like HepG2 or Jurkat cells where TMEM143 expression has been confirmed .

How should TMEM143 antibodies be stored for optimal stability?

For maximum stability and activity retention, TMEM143 antibodies should be stored at -20°C . Under these conditions, the antibodies remain stable for approximately one year after shipment. The typical storage buffer consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Importantly, aliquoting is generally unnecessary for -20°C storage, which simplifies laboratory management. Some commercial preparations (typically in 20μl sizes) may contain 0.1% BSA as a stabilizer .

What are the optimal antigen retrieval methods for TMEM143 immunohistochemistry?

For successful immunohistochemical detection of TMEM143:

• Primary recommended method: Antigen retrieval with TE buffer at pH 9.0

• Alternative method: Citrate buffer at pH 6.0

The alkaline pH method (TE buffer pH 9.0) typically provides better epitope exposure for many transmembrane proteins by more effectively breaking protein cross-links formed during fixation. For challenging tissue samples, extending the antigen retrieval time or testing both methods in parallel may help optimize signal-to-noise ratio. Human placenta tissue has been verified as a positive control for TMEM143 IHC applications .

How can I validate the specificity of a TMEM143 antibody?

A rigorous validation approach should include:

• Multiple application testing: Confirm consistent results across Western blot, IHC and other intended applications

• Positive controls: Use cell lines with confirmed TMEM143 expression (HH, HepG2, Jurkat, or Ramos cells)

• Band size verification: Confirm the presence of bands at the expected 45-52 kDa range

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to demonstrate signal reduction

• Knockout/knockdown validation: If possible, test in TMEM143 knockout/knockdown samples to confirm absence of signal

• Cross-reactivity assessment: Test on samples from other species if relevant to research

This multifaceted approach provides robust evidence of antibody specificity before proceeding with critical experiments.

What are the recommended protocols for Western blotting with TMEM143 antibodies?

For optimal Western blot results when detecting TMEM143:

• Sample preparation: Include protease inhibitors during cell/tissue lysis to prevent protein degradation

• Protein loading: Load 20-40 μg of total protein per lane

• Gel percentage: Use 10-12% SDS-PAGE gels for optimal resolution in the 45-52 kDa range

• Transfer conditions: Semi-dry or wet transfer at 100V for 60-90 minutes

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Dilute TMEM143 antibody 1:500-1:1000 in blocking buffer

• Incubation: Overnight at 4°C with gentle agitation

• Detection system: HRP-conjugated secondary antibody with ECL detection

Following this methodology helps ensure consistent and specific detection of TMEM143 protein in Western blot applications.

How can TMEM143 antibodies be used to investigate mitochondrial function?

As TMEM143 is localized to mitochondria , its antibodies can be valuable tools for investigating mitochondrial biology:

• Co-localization studies: Combine TMEM143 antibodies with known mitochondrial markers (e.g., MitoTracker, TOMM20) to study spatial distribution

• Mitochondrial fractionation: Use TMEM143 as a marker to validate mitochondrial isolation protocols

• Stress response: Examine TMEM143 expression changes under mitochondrial stress conditions (e.g., oxidative stress, ETC inhibitors)

• Mitophagy research: Investigate potential changes in TMEM143 during mitochondrial quality control processes

• Protein interaction studies: Use co-immunoprecipitation with TMEM143 antibodies to identify interaction partners within the mitochondrial membrane

These approaches can provide insights into the functional role of TMEM143 in mitochondrial biology and potentially reveal connections to cellular pathways affected in disease states.

What strategies can be employed to study TMEM143 in cancer research models?

Given the connection between TMEM family proteins and cancer , researchers can utilize TMEM143 antibodies in several advanced cancer research applications:

• Expression profiling: Compare TMEM143 levels across cancer cell lines and matched normal tissues

• Tissue microarray analysis: Perform IHC on cancer tissue microarrays to correlate expression with clinical parameters

• Functional studies: Combine antibody detection with knockdown/overexpression models to assess phenotypic changes

• Signaling pathway investigation: Examine relationships between TMEM143 and known cancer signaling pathways

• Drug response monitoring: Track TMEM143 expression changes in response to therapeutic agents

• Extracellular vesicle research: Investigate TMEM143 presence in cancer-derived exosomes or microvesicles

These approaches can help elucidate potential roles of TMEM143 in cancer development, progression, or treatment response.

What considerations are important when developing a multiplex immunofluorescence panel including TMEM143?

When incorporating TMEM143 antibodies into multiplex immunofluorescence panels:

• Antibody species selection: Choose primary antibodies raised in different host species to avoid cross-reactivity

• Epitope compatibility: Ensure that antibodies target non-overlapping epitopes when studying multiple aspects of TMEM143

• Fluorophore selection: Consider spectral overlap and design panels with sufficient separation between fluorophores

• Signal strength balancing: Adjust antibody concentrations to balance signals from high and low-abundance targets

• Fixation optimization: Test different fixation protocols as they can differentially affect epitope preservation

• Sequential staining: For challenging combinations, consider sequential staining with stripping steps

• Validation controls: Include single-stain controls to verify specificity in the multiplex context

These considerations help ensure reliable and interpretable results when studying TMEM143 alongside other proteins of interest.

How can I address weak or absent signal in TMEM143 Western blots?

When facing detection challenges in TMEM143 Western blots:

These systematic approaches help troubleshoot common issues encountered when detecting TMEM143 in Western blot applications.

What are common pitfalls in TMEM143 immunohistochemistry and how can they be addressed?

Common challenges in TMEM143 IHC applications include:

• Weak or variable staining:

  • Optimize antigen retrieval with TE buffer pH 9.0 as recommended

  • Increase antibody concentration (within 1:50-1:500 range)

  • Extend incubation time to overnight at 4°C

  • Use signal amplification systems like tyramide signal amplification

• Nonspecific background:

  • Implement additional blocking steps (avidin/biotin block if using biotin-based detection)

  • Extend washing steps and increase wash buffer volume

  • Pre-absorb antibodies with tissue powder

  • Include appropriate controls to distinguish specific from nonspecific signals

• Tissue-specific challenges:

  • For highly autofluorescent tissues, use Sudan Black B treatment

  • For high endogenous peroxidase activity, extend hydrogen peroxide blocking

  • For tissues with high fat content, optimize deparaffinization and clearing steps

These strategies can significantly improve TMEM143 detection quality and reproducibility in tissue sections.

How can cross-reactivity issues with TMEM143 antibodies be identified and mitigated?

To address potential cross-reactivity concerns:

• Identification methods:

  • Observe unexpected band patterns on Western blots

  • Note inconsistent staining patterns between different antibody clones

  • Compare results with antibodies targeting different epitopes of TMEM143

  • Validate using genetic knockdown/knockout approaches

• Mitigation strategies:

  • Select antibodies with extensively validated specificity data

  • Use antibodies with more specific epitope targets (C-terminal specific antibodies may offer higher specificity)

  • Increase stringency of washing conditions

  • Perform pre-absorption with related proteins

  • Consider using monoclonal antibodies for highly specific applications

• Validation approaches:

  • Peptide competition assays

  • Immunoprecipitation followed by mass spectrometry

  • Parallel testing with multiple TMEM143 antibodies targeting different epitopes

Implementing these approaches helps ensure experimental results accurately reflect TMEM143 biology rather than artifact signals.

How can TMEM143 antibodies be utilized in proximity ligation assays to study protein-protein interactions?

Proximity Ligation Assay (PLA) is a powerful technique for studying TMEM143 interactions:

• Experimental design considerations:

  • Combine TMEM143 antibody with antibodies against suspected interaction partners

  • Ensure antibodies are raised in different species for proper secondary antibody recognition

  • For mitochondrial interactions, include appropriate membrane permeabilization steps

  • Consider fixation methods that preserve membrane protein conformations

• Protocol optimization:

  • Test multiple antibody dilutions to maximize signal-to-noise ratio

  • Optimize permeabilization to ensure access to mitochondrial membranes

  • Include appropriate controls (positive interaction controls and negative antibody controls)

  • Consider subcellular fractionation before PLA to enrich for mitochondrial proteins

• Data interpretation:

  • Quantify PLA signals in relation to subcellular markers to confirm mitochondrial localization

  • Use Z-stack imaging to capture the full 3D distribution of interaction signals

  • Implement computational analysis to quantify interaction frequency and spatial distribution

This approach can reveal novel TMEM143 interaction networks and provide insights into its functional roles in mitochondria.

What strategies can be employed to study post-translational modifications of TMEM143?

To investigate post-translational modifications (PTMs) of TMEM143:

• Combined immunoprecipitation approach:

  • Use TMEM143 antibodies for immunoprecipitation

  • Probe with PTM-specific antibodies (phospho, ubiquitin, SUMO, acetylation)

  • Confirm with mass spectrometry analysis

• Modification-specific detection:

  • Treat samples with phosphatases, deubiquitinases, or other modification-removing enzymes

  • Observe mobility shifts in Western blots as indicators of modifications

  • Use Phos-tag gels for enhanced separation of phosphorylated species

• Site-specific investigations:

  • Generate phospho-specific or other PTM-specific antibodies for key modification sites

  • Use site-directed mutagenesis of predicted modification sites to assess functional impact

  • Employ cell treatments that alter modification states (kinase inhibitors, proteasome inhibitors)

These approaches can reveal regulatory mechanisms controlling TMEM143 function, localization, or stability within mitochondrial membranes.

How can single-cell analysis techniques be combined with TMEM143 antibodies to study heterogeneity in expression patterns?

Integrating TMEM143 antibody detection with single-cell technologies:

• Single-cell Western blotting:

  • Optimize TMEM143 antibody dilutions for microfluidic single-cell Western platforms

  • Calibrate detection sensitivity for the expected expression range

  • Include co-detection of cell type markers to correlate with TMEM143 expression

• Mass cytometry (CyTOF):

  • Develop metal-conjugated TMEM143 antibodies

  • Create panels combining TMEM143 with mitochondrial markers and cell state indicators

  • Implement permeabilization protocols optimized for mitochondrial proteins

• Imaging mass cytometry/CODEX:

  • Apply TMEM143 antibodies in multiplexed tissue imaging platforms

  • Co-stain with spatial references to map expression to tissue microenvironments

  • Develop computational workflows to quantify cell-to-cell variation in expression

• Single-cell RNA-protein correlation:

  • Combine TMEM143 protein detection with RNA sequencing in platforms like CITE-seq

  • Analyze correlation between transcript and protein levels to identify post-transcriptional regulation

These advanced techniques provide unprecedented insights into cellular heterogeneity of TMEM143 expression and its relationship to cell state, tissue context, and disease progression.