TMEM43 Antibody, HRP conjugated

What is TMEM43 and what are its primary cellular functions?

TMEM43, also known as Transmembrane Protein 43 or LUMA, is a protein that contributes to the physical and functional stability of the nuclear envelope by organizing protein complexes at the inner nuclear membrane . Recent research has characterized TMEM43 as a novel ion channel permeable to Na+, K+, and Cs+ ions, functioning as a nonselective cation channel that responds to external pH levels . Beyond structural roles, TMEM43 is involved in retaining emerin at the inner nuclear membrane, modulating innate immune signaling through the cGAS-STING pathway by interacting with RNF26, and mediating NF-κB activation by acting downstream of EGFR and upstream of CARD10 . It also contributes to passive conductance current in cochlear glia-like supporting cells through gap junctions, which is necessary for hearing and speech discrimination .

What are the key differences between various TMEM43 antibody formats and what factors should guide selection for experimental design?

When selecting a TMEM43 antibody, researchers should consider several factors including host species, clonality, conjugation, and binding specificity. Rabbit-derived antibodies are common for TMEM43 detection and are available in both polyclonal and monoclonal formats . Monoclonal antibodies like EPR15378(B) offer higher specificity and reproducibility, while polyclonal antibodies may provide stronger signals by recognizing multiple epitopes .

For binding specificity, antibodies targeting different amino acid regions (such as AA 80-310, AA 178-312, or AA 195-223) may have different detection capabilities depending on protein conformation and experimental conditions . HRP-conjugated antibodies are particularly suitable for direct detection in assays like ELISA and Western blot, eliminating the need for secondary antibodies . The selection should be guided by application requirements, with consideration of validated applications listed in product documentation (WB, IHC-P, ELISA) and species reactivity (human, mouse, rat) .

How does TMEM43 localization influence antibody selection and experimental design?

When designing experiments, researchers should select antibodies validated for the specific cellular compartment of interest. For nuclear envelope studies, antibodies recognizing domains accessible in this context are essential. For cytoplasmic detection, as in cancer research, antibodies validated in IHC with appropriate permeabilization protocols should be used . The experimental design should incorporate proper cell fixation and permeabilization methods to ensure antibody access to the target protein in its native location. Additionally, co-localization studies with known markers of specific cellular compartments can validate the observed TMEM43 distribution patterns and provide context for functional analyses .

What are the optimal protocols for using HRP-conjugated TMEM43 antibodies in Western blot applications?

For Western blot applications using HRP-conjugated TMEM43 antibodies, the following optimized protocol is recommended:

• Sample Preparation: Prepare tissue or cell lysates with complete protease inhibitors. For nuclear envelope proteins like TMEM43, specialized lysis buffers containing mild detergents are recommended.

• Protein Quantification: Standardize protein loading using Bradford or BCA assay.

• Gel Electrophoresis: Separate 20-30 μg protein on 10-12% SDS-PAGE gels (TMEM43 molecular weight ~43 kDa).

• Transfer: Use PVDF membrane for optimal protein binding with transfer at 100V for 60-90 minutes.

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

• Primary Antibody Incubation: Dilute HRP-conjugated TMEM43 antibody at 1:5000 ratio (based on the dilution used in successful applications ). Incubate overnight at 4°C.

• Washing: Perform 5 washes with TBST, 5 minutes each.

• Detection: Apply ECL substrate directly and image membrane. No secondary antibody is needed due to HRP conjugation.

• Quantification: Normalize TMEM43 signal to housekeeping proteins like GAPDH or β-actin.

This protocol has been validated in multiple studies, including those demonstrating increased TMEM43 expression in hepatocellular carcinoma compared to adjacent tissues .

How can researchers optimize immunohistochemistry protocols when using HRP-conjugated TMEM43 antibodies for tissue samples?

Optimizing immunohistochemistry protocols for HRP-conjugated TMEM43 antibodies requires specific considerations:

• Tissue Preparation: Use formalin-fixed, paraffin-embedded (FFPE) sections at 4-6 μm thickness. Fresh frozen sections may provide stronger signal but poorer morphology.

• Antigen Retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes is typically effective for TMEM43 detection.

• Endogenous Peroxidase Blocking: Treat with 3% hydrogen peroxide for 10 minutes to prevent background from endogenous peroxidases.

• Antibody Dilution: For HRP-conjugated antibodies, a dilution range of 1:20-1:200 is recommended based on application notes . Begin with 1:50 and optimize if needed.

• Incubation Parameters: Incubate primary antibody overnight at 4°C in a humidified chamber.

• Signal Amplification: If signal strength is inadequate, consider polymer-based detection systems compatible with HRP-conjugated antibodies.

• Counterstaining: Use hematoxylin for nuclear counterstaining, being careful not to obscure cytoplasmic TMEM43 staining.

• Controls: Include both positive controls (known TMEM43-expressing tissues like spleen) and negative controls (antibody diluent only).

• Scoring System: Develop a standardized scoring system for TMEM43 expression (e.g., intensity scale 0-3 and percentage of positive cells).

This approach has been validated in tissue microarray studies showing predominant cytoplasmic localization of TMEM43 in hepatocellular carcinoma tissues .

What considerations are important when designing ELISA protocols using HRP-conjugated TMEM43 antibodies?

When designing ELISA protocols with HRP-conjugated TMEM43 antibodies, several critical considerations must be addressed:

• Assay Format: For TMEM43 detection, a sandwich ELISA using a capture antibody against a different epitope than the HRP-conjugated detection antibody provides optimal specificity.

• Antibody Pairs: Validate compatible antibody pairs that do not compete for the same epitope. For example, pair an antibody targeting AA 80-310 with one targeting AA 195-223 .

• Sample Preparation: Optimize protein extraction methods based on TMEM43 localization. For membrane proteins, detergent-based lysis buffers containing 0.5-1% Triton X-100 are typically effective.

• Standard Curve: Develop a standard curve using recombinant TMEM43 protein spanning the amino acid region recognized by the antibody (e.g., 80-310AA) .

• Blocking Optimization: Test various blocking agents (BSA, casein, commercial blocking buffers) to minimize background while preserving specific signal.

• Antibody Concentration: For HRP-conjugated detection antibodies, begin with a 1:100 dilution and optimize through serial dilutions.

• Substrate Selection: TMB (3,3',5,5'-Tetramethylbenzidine) offers high sensitivity with HRP conjugates and allows for quantifiable colorimetric readout.

• Signal Development Time: Optimize substrate incubation time to achieve maximum signal-to-noise ratio, typically 15-30 minutes.

• Plate Type: Use high-binding ELISA plates to ensure efficient protein capture.

These considerations have been validated in experimental applications using TMEM43 antibodies with ELISA compatibility .

What are common issues with HRP-conjugated TMEM43 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with HRP-conjugated TMEM43 antibodies:

• High Background Signal

  • Problem: Non-specific binding causing widespread staining

  • Solution: Increase blocking time/concentration, optimize antibody dilution (start at 1:5000 for WB ), add 0.1% Tween-20 to wash buffers, and ensure proper blocking agent selection

• Weak or Absent Signal

  • Problem: Insufficient antigen exposure or antibody concentration

  • Solution: Optimize antigen retrieval methods, increase antibody concentration, extend incubation time, verify protein expression in positive control samples like human spleen tissue

• Multiple Bands in Western Blot

  • Problem: Non-specific binding or TMEM43 isoforms/post-translational modifications

  • Solution: Increase antibody dilution, optimize washing steps, verify with knockout/knockdown controls, consider alternative antibody targeting different epitope region

• Degraded HRP Activity

  • Problem: Reduced enzyme activity affecting signal generation

  • Solution: Avoid freeze-thaw cycles, store antibody according to manufacturer recommendations (typically with 50% glycerol at -20°C ), prepare working dilutions fresh

• Inconsistent Results Between Experiments

  • Problem: Protocol variations or reagent degradation

  • Solution: Standardize protocols, use consistent positive controls, prepare aliquots of antibody to avoid repeated freeze-thaw cycles

Each troubleshooting approach should be validated with appropriate controls, including tissues known to express TMEM43 at high levels, such as spleen or placenta .

How can researchers validate the specificity of HRP-conjugated TMEM43 antibodies in their experimental systems?

Validating the specificity of HRP-conjugated TMEM43 antibodies is crucial for ensuring reliable research outcomes. Multiple complementary approaches should be employed:

• Genetic Controls

  • Perform parallel experiments using TMEM43 knockdown or knockout models

  • Compare antibody signals between wild-type and TMEM43-deficient samples

  • Expected outcome: Significantly reduced or absent signal in TMEM43-deficient samples

• Peptide Competition Assay

  • Pre-incubate antibody with excess immunizing peptide (e.g., recombinant TMEM43 protein fragments spanning AA 80-310)

  • Run parallel Western blots or IHC with blocked and unblocked antibody

  • Expected outcome: Signal should be significantly reduced with peptide-blocked antibody

• Multiple Antibody Validation

  • Compare staining patterns using different TMEM43 antibodies targeting distinct epitopes

  • Concordant results across antibodies support specificity

• Subcellular Localization Assessment

  • Verify that detected TMEM43 shows expected localization patterns (nuclear envelope, cytoplasm)

  • Perform co-localization studies with established nuclear envelope or cytoplasmic markers

• Molecular Weight Verification

  • Confirm that detected bands in Western blot match the predicted molecular weight of TMEM43 (~43 kDa)

  • Assess whether band pattern matches known isoforms or post-translational modifications

• Positive and Negative Tissue Controls

  • Include tissues known to express high levels of TMEM43 (spleen, placenta)

  • Include tissues with minimal TMEM43 expression as negative controls

These validation approaches have been successfully applied in studies examining TMEM43 expression in hepatocellular carcinoma and other tissues .

How can TMEM43 antibodies be utilized to investigate its role as a novel ion channel in electrophysiological studies?

The recent characterization of TMEM43 as a nonselective cation channel with pH sensitivity opens new research directions requiring specialized antibody applications:

• Functional Blocking Studies:

  • Apply non-conjugated TMEM43 antibodies targeting extracellular domains during patch-clamp recordings

  • Monitor changes in channel conductance to Na+, K+, and Cs+ ions

  • Compare results with pharmacological channel blockers to confirm specificity

• Structure-Function Analysis:

  • Use site-specific antibodies targeting different domains (particularly TM3 and Loop2, which contain pore-forming residues)

  • Correlate antibody binding with altered channel function through patch-clamp measurements

  • Design experiments to investigate pH sensitivity (as TMEM43 current decreases with lowering external pH)

• Co-immunoprecipitation Studies:

  • Use TMEM43 antibodies for pull-down experiments followed by mass spectrometry

  • Identify potential channel regulatory partners or subunits

  • Validate interactions with known channel modulators

• Channel Expression and Trafficking:

  • Employ antibodies in surface biotinylation assays to quantify membrane-localized TMEM43

  • Track channel internalization and recycling using antibody-based endocytosis assays

  • Correlate expression levels with electrophysiological recordings of channel activity

• Single-Channel Recording Correlation:

  • Following observation of stochastic channel openings in lipid bilayer-reconstituted purified TMEM43

  • Use antibodies to confirm identity of recorded channels

  • Correlate single-channel properties with whole-cell currents in heterologous expression systems

These approaches can help elucidate how TMEM43 functions as an ion channel and how disease-associated mutations, such as p.(Arg372Ter), lead to loss of channel activity in a dominant-negative fashion .

What approaches are recommended for investigating TMEM43's involvement in cancer progression using HRP-conjugated antibodies?

TMEM43's emerging role in cancer progression, particularly in hepatocellular carcinoma and glioblastoma , necessitates specialized antibody applications:

• Tissue Microarray Analysis:

  • Systematically evaluate TMEM43 expression across tumor grades and stages using HRP-conjugated antibodies

  • Develop quantitative scoring systems for cytoplasmic TMEM43 expression

  • Correlate expression patterns with patient survival and clinical parameters

  • Example finding: TMEM43 is highly expressed in 68 out of 96 HCC tissues, with primarily cytoplasmic localization

• Cellular Signaling Pathway Analysis:

  • Use antibodies to track TMEM43's involvement in:

    • NF-κB activation pathways

    • EGFR downstream signaling

    • CARD10-associated signaling events

  • Apply phospho-specific antibodies against pathway components with and without TMEM43 manipulation

• Protein-Protein Interaction Studies:

  • Employ co-immunoprecipitation with TMEM43 antibodies followed by Western blot analysis

  • Validate interactions with reported partners (VDAC1, USP7)

  • Investigate deubiquitination effects on TMEM43 stability and function

• Functional Consequence Assessment:

  • Combine antibody-based detection with functional assays:

    • Cell viability (CCK-8)

    • Proliferation (cloning assays)

    • Migration (Transwell)

    • Apoptosis (flow cytometry)

  • Correlate protein expression levels with phenotypic outcomes

• In vivo Tumor Model Analysis:

  • Apply IHC with HRP-conjugated TMEM43 antibodies to xenograft tissues

  • Track expression changes during tumor progression

  • Correlate with metastatic potential and treatment response

These approaches can help elucidate how TMEM43 contributes to cancer progression and potentially identify it as a therapeutic target or biomarker .

How can researchers design experiments to investigate the relationship between TMEM43 mutations and disease phenotypes?

Investigating the relationship between TMEM43 mutations and disease phenotypes requires carefully designed experimental approaches:

• Mutation-Specific Antibody Application:

  • Develop or obtain antibodies that differentially recognize wild-type versus mutant TMEM43

  • Focus on clinically relevant mutations:

    • p.(Ser358Leu) associated with arrhythmogenic right ventricular cardiomyopathy (ARVC)

    • p.(Arg372Ter) associated with hearing loss

  • Use these antibodies to track mutant protein localization and expression levels

• Functional Comparative Analysis:

  • Apply wild-type and mutant-specific antibodies in parallel experiments

  • Compare:

    • Subcellular localization patterns

    • Protein-protein interactions

    • Channel function (for ion channel activity)

    • Nuclear envelope integrity (for structural roles)

  • Example finding: p.(Arg372Ter) mutation results in loss of channel activity in a dominant-negative fashion

• Disease Model Validation:

  • Use HRP-conjugated antibodies in tissue samples from:

    • Patient-derived samples (when available)

    • Animal models expressing TMEM43 mutations

    • Cell models with CRISPR/Cas9-engineered mutations

  • Correlate antibody-detected expression patterns with phenotypic manifestations

• Protein Stability and Processing Analysis:

  • Track mutant protein half-life compared to wild-type

  • Investigate whether mutations alter post-translational modifications

  • Examine potential degradation pathways using inhibitors of protein degradation

• Interaction Partner Profiling:

  • Compare binding profiles of wild-type versus mutant TMEM43:

    • Interaction with emerin and lamins in nuclear envelope

    • Association with gap junction proteins like Connexin 26 and 30

    • Binding to K+ channels like KCNK3 (TASK-1)

  • Identify differential protein interactions that may explain disease mechanisms

These approaches can help elucidate how specific TMEM43 mutations disrupt normal function and lead to diverse disease phenotypes ranging from cardiomyopathy to hearing loss .

How should researchers interpret variations in TMEM43 expression patterns across different tissue types and disease states?

Interpreting TMEM43 expression variations requires systematic analysis and consideration of multiple factors:

• Baseline Expression Profiling:

  • Establish normal TMEM43 expression patterns across tissue types

  • Consider tissue-specific roles:

    • Nuclear envelope stability in most cell types

    • Ion channel function in specialized cells (e.g., cochlear supporting cells)

    • Signaling pathway involvement in specific contexts

  • Use standardized quantification methods (Western blot band intensity, IHC H-scores)

• Disease-Associated Changes:

  • Compare expression in matched disease and normal tissues

  • Example finding: TMEM43 is significantly increased in hepatocellular carcinoma compared to adjacent normal tissue

  • Consider both expression level and localization changes:

    • Cytoplasmic accumulation in HCC

    • Altered nuclear envelope distribution in cardiomyopathy

• Context-Dependent Function Analysis:

  • Recognize that TMEM43 may have different functions in different cellular contexts

  • Correlate expression patterns with:

    • Cell type-specific interaction partners

    • Downstream signaling pathway activation

    • Cellular phenotypes (proliferation, migration, etc.)

• Mutation Impact Assessment:

  • Differentiate between expression level changes and functional alterations

  • Example: p.(Arg372Ter) mutation causes loss of channel activity despite protein expression

  • Consider dominant-negative effects of mutant proteins on wild-type function

• Multi-Method Validation:

  • Confirm expression changes using complementary techniques:

    • Protein detection (Western blot, IHC)

    • mRNA analysis (qPCR, RNA-seq)

    • Functional assays (electrophysiology, migration, etc.)

By systematically analyzing TMEM43 expression variations through these approaches, researchers can gain insights into its diverse physiological roles and pathological implications across different tissues and disease states .

What methodological approaches are recommended for studying TMEM43's interactions with other proteins using antibody-based techniques?

Studying TMEM43's protein interactions requires specialized antibody-based approaches:

• Co-Immunoprecipitation (Co-IP) Optimization:

  • Use non-conjugated TMEM43 antibodies for pull-down experiments

  • Optimize lysis conditions to preserve membrane protein interactions:

    • Mild detergents (0.5-1% NP-40 or digitonin)

    • Physiological salt concentrations

    • Protease and phosphatase inhibitors

  • Validate interactions with reported partners:

    • Nuclear envelope proteins (emerin, lamins)

    • Gap junction proteins (Connexin 26, Connexin 30)

    • Signaling molecules (CARMA3, VDAC1, USP7)

• Proximity Ligation Assay (PLA):

  • Detect in situ protein interactions with spatial resolution

  • Combine TMEM43 antibodies with antibodies against suspected interaction partners

  • Quantify interaction signals in different subcellular compartments

  • Particularly useful for membrane-associated interactions that may be disrupted during extraction

• FRET-Based Interaction Analysis:

  • Use fluorophore-conjugated antibodies for Förster Resonance Energy Transfer

  • Detect close-proximity interactions (<10 nm)

  • Apply in fixed cells to preserve native interaction contexts

• Crosslinking Immunoprecipitation:

  • Apply membrane-permeable crosslinkers to stabilize transient interactions

  • Use TMEM43 antibodies for subsequent immunoprecipitation

  • Identify interaction partners by mass spectrometry

  • Particularly valuable for capturing weak or transient interactions

• Domain-Specific Interaction Mapping:

  • Use antibodies targeting specific TMEM43 domains (e.g., AA 80-310, AA 178-312)

  • Determine which domains are critical for specific protein interactions

  • Correlate with functional studies examining channel activity or structural roles

These methodological approaches have successfully identified TMEM43 interactions with proteins involved in diverse cellular processes, from nuclear envelope stability to signaling pathway activation and ion channel function .

How can researchers integrate TMEM43 antibody-based findings with other omics data to build comprehensive disease models?

Integrating TMEM43 antibody-based findings with multi-omics data requires systematic analytical approaches:

• Correlative Transcriptomics Integration:

  • Compare protein expression (antibody-based) with mRNA levels (RNA-seq)

  • Identify concordant or discordant expression patterns suggesting post-transcriptional regulation

  • Example application: RNA-seq data from TCGA database revealed TMEM43 upregulation in HCC, which was confirmed at protein level by Western blot

  • Analyze co-expression networks to identify functionally related gene clusters

• Proteomics Data Integration:

  • Combine antibody-detected TMEM43 levels with global proteomics data

  • Identify co-regulated protein networks

  • Examine post-translational modifications affecting TMEM43 function

  • Example focus: Investigate deubiquitination effects of USP7 on TMEM43 stability

• Functional Genomics Correlation:

  • Integrate antibody-based protein detection with functional genomics screens

  • Correlate TMEM43 expression with phenotypic outcomes in CRISPR screens

  • Example application: Correlate TMEM43 expression with cell viability, proliferation, migration, and apoptosis measured through functional assays

• Clinical Data Integration:

  • Correlate TMEM43 expression patterns with patient outcomes

  • Develop predictive models combining molecular and clinical features

  • Example finding: High TMEM43 expression in HCC correlates with poor prognosis

• Pathway Analysis Enhancement:

  • Map antibody-detected TMEM43 interactions to signaling pathways

  • Integrate with phosphoproteomics data to assess pathway activation

  • Example pathways:

    • NF-κB signaling downstream of EGFR

    • cGAS-STING pathway in innate immunity

    • Gap junction-mediated signaling in cochlear cells

• Multi-Modal Data Visualization:

  • Develop integrated visualizations combining:

    • Protein expression (antibody-based)

    • mRNA levels (transcriptomics)

    • Mutation data (genomics)

    • Clinical parameters

  • Use dimensionality reduction approaches to identify disease subtypes