TMEM43 Antibody, Biotin conjugated

What is TMEM43 and why is it a significant research target?

TMEM43 (Transmembrane Protein 43) is a four-transmembrane protein primarily localized to the endoplasmic reticulum (ER) in its wild-type form. It has gained significant research interest due to its association with arrhythmogenic cardiomyopathy (ACM), particularly through the p.S358L variant, which causes a fully penetrant heart disease resulting in impaired cardiac function or fatal arrhythmia . The protein has a calculated molecular weight of approximately 44,876 Da , and variants have also been implicated in auditory disorders through mutations such as p.R372X . As a membrane protein with multiple transmembrane domains, TMEM43 research requires specialized antibodies for proper detection and characterization in various experimental contexts.

What are the specific advantages of using biotin-conjugated TMEM43 antibodies?

Biotin-conjugated TMEM43 antibodies offer several methodological advantages for research applications:

• Enhanced signal amplification: The biotin-streptavidin system provides one of the strongest non-covalent biological interactions (Kd ≈ 10^-15 M), allowing for significant signal enhancement in detection systems.

• Versatile detection options: Biotin-conjugated antibodies can be paired with multiple detection reagents including streptavidin-HRP, streptavidin-fluorophores, or streptavidin-gold particles, providing flexibility across different experimental platforms.

• Effective for low-abundance targets: When TMEM43 expression is limited, the signal amplification properties of biotin-conjugated antibodies can improve detection sensitivity, particularly in ELISA applications .

• Reduced background interference: The biotin-streptavidin system often produces cleaner results with less non-specific binding compared to traditional secondary antibody detection methods.

• Stability during storage: Biotin conjugation typically enhances antibody stability during storage compared to some other conjugation methods.

How do I select the appropriate TMEM43 antibody for my specific application?

When selecting a TMEM43 antibody for research, consider these methodological factors:

Target epitope considerations:

• For studying wild-type TMEM43, antibodies targeting conserved regions (such as AA 80-310) provide reliable detection across experimental systems .

• For variant-specific research (e.g., S358L variant in cardiomyopathy studies), select antibodies that can recognize the specific altered epitope region.

Species reactivity requirements:

• Available antibodies demonstrate reactivity to human and mouse TMEM43 , which is crucial for translational research between model systems and human samples.

• Cross-check the validated species reactivity in the datasheet against your experimental model.

Application compatibility:

• For Western Blotting: Polyclonal antibodies against TMEM43 have been validated at dilutions of 1:500-1:2000 .

• For ELISA: Biotin-conjugated antibodies targeting AA 178-312 have demonstrated effectiveness .

• For Immunohistochemistry: Several TMEM43 antibodies have been validated for IHC applications, with specific recommendations for paraffin sections .

Clonality selection:

• Polyclonal antibodies provide broad epitope recognition, beneficial for applications like immunoprecipitation or initial characterization studies.

• Monoclonal antibodies offer higher consistency between lots and more specific epitope targeting.

What is the optimal protocol for using biotin-conjugated TMEM43 antibodies in ELISA?

The following protocol optimizes ELISA performance with biotin-conjugated TMEM43 antibodies:

Materials required:

• Biotin-conjugated anti-TMEM43 antibody targeting AA 178-312

• High-binding ELISA microplate

• Blocking buffer (typically 3% BSA in PBS)

• Streptavidin-HRP conjugate

• TMB substrate solution

• Stop solution (2N H₂SO₄)

• Wash buffer (PBS with 0.05% Tween-20)

Protocol:

• Antigen coating: Coat wells with recombinant TMEM43 protein or cell lysate containing TMEM43 at 1-10 μg/ml in carbonate buffer (pH 9.6). Incubate overnight at 4°C.

• Blocking: Block non-specific binding sites with 3% BSA in PBS for 1 hour at room temperature.

• Primary antibody: Apply the biotin-conjugated TMEM43 antibody at 1:500-1:2000 dilution in antibody diluent. Incubate for 2 hours at room temperature or overnight at 4°C.

• Detection system: Add streptavidin-HRP at manufacturer's recommended dilution (typically 1:2000-1:5000). Incubate for 30-60 minutes at room temperature.

• Signal development: Add TMB substrate and monitor color development. Stop the reaction with stop solution when appropriate signal intensity is achieved.

• Analysis: Read absorbance at 450 nm with 620 nm as reference wavelength.

Critical optimization points:

• When detecting low-abundance TMEM43, extend primary antibody incubation to overnight at 4°C.

• Include negative controls (omitting primary antibody) to assess background.

• For quantitative analysis, establish a standard curve using recombinant TMEM43 protein.

How should biotin-conjugated TMEM43 antibodies be stored to maintain optimal activity?

Proper storage is crucial for maintaining the functional integrity of biotin-conjugated TMEM43 antibodies:

Long-term storage recommendations:

• Store at -20°C for up to one year in small aliquots to minimize freeze-thaw cycles .

• Ensure antibody is stored in a buffer containing a cryoprotectant such as 50% glycerol.

Short-term storage:

• For frequent use within a one-month period, store at 4°C .

• Protect from light, especially if the antibody contains both biotin and a fluorophore.

Handling precautions:

• Avoid repeated freeze-thaw cycles, which can cause biotin conjugate degradation and reduced antibody affinity.

• When thawing, allow the antibody to come to room temperature completely before opening the vial to prevent condensation.

• Centrifuge briefly before opening to collect solution at the bottom of the tube.

Working solution preparation:

• Prepare fresh working dilutions on the day of the experiment.

• If working dilutions must be stored, keep at 4°C for no more than 24 hours.

• Include 0.02% sodium azide in storage buffer as a preservative, but note that this may inhibit HRP activity in downstream applications.

How can biotin-conjugated TMEM43 antibodies be optimized for studying the S358L variant associated with arrhythmogenic cardiomyopathy?

The TMEM43 S358L variant demonstrates altered cellular localization and post-translational modifications that require specialized experimental approaches :

Variant-specific detection strategy:

• Epitope accessibility assessment: The S358L variant accumulates at the nuclear envelope rather than the ER , potentially altering epitope accessibility. Test multiple antibodies targeting different regions of TMEM43 to identify those that maintain reactivity to the mislocalized variant.

• Glycosylation-aware methodology: Since TMEM43 S358L undergoes increased N-linked glycosylation , implement a dual approach:

  • Use biotin-conjugated antibodies for direct detection

  • Complement with deglycosylation experiments (PNGase F treatment) to confirm identity of bands in Western blotting

• Sub-cellular fractionation protocol optimization:

  • Nuclear envelope fraction: 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 1 mM DTT, protease inhibitors

  • Perform differential centrifugation at 1,300 × g for nuclear fraction followed by additional purification steps

  • Validate fractionation using markers such as Lamin B1 (nuclear envelope) and Calnexin (ER)

• Immunofluorescence co-localization workflow:

  • Fix cells in 4% paraformaldehyde for 15 minutes

  • Permeabilize with 0.2% Triton X-100 for 10 minutes

  • Use biotin-conjugated TMEM43 antibody with streptavidin-fluorophore for detection

  • Co-stain with nuclear envelope markers (Lamin A/C) and ER markers (Calnexin)

  • Analyze using confocal microscopy with Z-stack acquisition

What controls are essential when using biotin-conjugated TMEM43 antibodies for immunoprecipitation studies?

For rigorous immunoprecipitation experiments with biotin-conjugated TMEM43 antibodies, the following controls are essential:

Experimental controls table:

Methodological considerations:

• For membrane proteins like TMEM43, use lysis buffers containing 1% NP-40 or 1% Triton X-100 with 0.1% SDS to effectively solubilize while maintaining protein-protein interactions.

• Consider crosslinking the biotin-conjugated antibody to streptavidin beads to prevent antibody co-elution and interference with detection.

• For co-immunoprecipitation studies investigating TMEM43 interaction partners, include targeted western blots for known ER-associated proteins as positive controls.

How can I design experiments to investigate TMEM43's role in transcriptional regulation using biotin-conjugated antibodies?

Research indicates TMEM43 variants affect transmural gene expression patterns in cardiomyopathy , suggesting a role in transcriptional regulation. The following experimental design leverages biotin-conjugated antibodies for this investigation:

Chromatin interaction analysis workflow:

• Proximity ligation assay (PLA) protocol:

  • Fix cells with 4% paraformaldehyde

  • Permeabilize with 0.3% Triton X-100

  • Block with 3% BSA

  • Incubate with biotin-conjugated TMEM43 antibody and antibodies against transcription factors or nuclear envelope proteins

  • Apply PLA probes and perform ligation and amplification according to manufacturer's protocol

  • Quantify interaction signals as puncta per nucleus

• ChIP-seq approach with proximity labeling:

  • Express BirA*-TMEM43 fusion protein in cardiomyocytes

  • Treat cells with biotin to label proteins in proximity

  • Perform chromatin immunoprecipitation using biotin-conjugated TMEM43 antibody

  • Sequence precipitated DNA to identify genomic regions associated with TMEM43

• Transmural expression gradient analysis:

  • Isolate cardiomyocytes from different myocardial layers (endocardium, midmyocardium, epicardium)

  • Perform immunofluorescence with biotin-conjugated TMEM43 antibody

  • Conduct RNA-seq on separated layers

  • Compare transmural gene expression patterns between wild-type and TMEM43 variant samples

Data analysis considerations:

• Utilize gene ontology analysis to identify pathways affected by TMEM43 variant expression

• Apply motif analysis to ChIP-seq data to identify potential transcription factor binding sites

• Correlate TMEM43 localization patterns with gene expression changes across the myocardial wall

What are common issues when using biotin-conjugated TMEM43 antibodies and how can they be resolved?

Troubleshooting guide for biotin-conjugated TMEM43 antibody applications:

Method-specific troubleshooting:

• For Western blotting issues with membrane proteins like TMEM43:

  • Ensure complete solubilization using buffer containing 1% SDS

  • Transfer high molecular weight and hydrophobic proteins using lower methanol concentration (5-10%) in transfer buffer

  • Extend transfer time or use semi-dry transfer systems

• For ELISA optimization:

  • If signal is low, implement a sandwich ELISA format with capture antibody against a different TMEM43 epitope

  • Test different blocking agents (BSA vs. casein vs. commercial blockers)

  • Optimize streptavidin-HRP concentration and incubation conditions

How do I interpret conflicting results between different detection methods when studying TMEM43?

When different methodologies yield apparently contradictory results regarding TMEM43 localization, expression, or function, consider these analytical approaches:

Methodology-dependent result interpretation framework:

• Reconciling localization discrepancies:

  • TMEM43 can exhibit different localizations depending on variant (WT predominantly in ER vs. S358L in nuclear envelope)

  • Fixation methods affect membrane protein epitope accessibility differently

  • Resolution limitations of different microscopy techniques impact apparent localization

  Resolution strategy: Perform super-resolution microscopy (STORM, PALM) with biotin-conjugated TMEM43 antibody and streptavidin-fluorophores to precisely map subcellular distribution.

• Expression level inconsistencies:

  • Western blot quantification may detect primarily unglycosylated forms

  • ELISA might preferentially detect certain epitopes based on accessibility

  • qPCR data reflects mRNA levels which may not correlate with protein levels

  Resolution strategy: Implement parallel detection of total protein (Western blot), mRNA (qPCR), and functional protein (activity assays) to build a complete picture.

• Functional role contradictions:

  • TMEM43's impact on gene expression may be context-dependent and vary across cell types

  • Acute vs. chronic effects may differ substantially

  • Direct vs. indirect effects may be difficult to distinguish

  Resolution strategy: Develop time-course experiments with inducible expression systems to distinguish immediate vs. adaptive responses to TMEM43 variants.

How can I quantitatively assess TMEM43 expression patterns across cardiac tissue sections using biotin-conjugated antibodies?

For quantitative assessment of TMEM43 expression across cardiac tissue sections, implement this systematic approach:

Quantitative immunohistochemistry protocol:

• Tissue preparation:

  • Fix cardiac tissue in 4% paraformaldehyde

  • Prepare consistent section thickness (5-7 μm)

  • Include sections representing all myocardial layers (endocardium to epicardium)

• Standardized staining procedure:

  • Perform heat-induced epitope retrieval with citrate buffer (pH 6.0)

  • Block endogenous biotin using avidin/biotin blocking kit

  • Apply biotin-conjugated TMEM43 antibody at optimized dilution

  • Detect with streptavidin-HRP and DAB or fluorescent streptavidin conjugate

  • Include DAPI counterstain for nuclear identification

• Quantitative image acquisition:

  • Capture images using standardized exposure settings

  • Include calibration controls in each session

  • Acquire z-stacks when using confocal microscopy

  • Maintain consistent magnification across samples

• Analysis workflow:

  • Use automated image analysis software (ImageJ/FIJI with appropriate plugins)

  • Segment tissue into defined transmural zones (endocardium, midmyocardium, epicardium)

  • Quantify signal intensity, localization pattern, and co-localization with compartment markers

  • Apply statistical methods to compare transmural gradients

Data representation:

• Generate heat maps of TMEM43 expression across the myocardial wall

• Create quantitative plots of signal intensity vs. distance from endocardium

• Compare wild-type patterns with those of TMEM43 variants

This approach enables detection of the altered transmural gene expression patterns observed in TMEM43-related cardiomyopathy , providing insights into pathophysiological mechanisms.

What emerging methodologies might enhance TMEM43 research using biotin-conjugated antibodies?

Several cutting-edge methodologies can be integrated with biotin-conjugated TMEM43 antibodies to advance understanding of this protein's function:

• Proximity labeling approaches:

  • BioID or TurboID fusion with TMEM43 to identify proximal interacting proteins

  • APEX2-based proximity labeling for temporal analysis of TMEM43 interaction networks

  • Integration with mass spectrometry for unbiased interactome analysis

• Super-resolution microscopy applications:

  • DNA-PAINT using biotin-conjugated primary antibodies for nanoscale resolution of TMEM43 localization

  • Expansion microscopy to physically enlarge samples for improved visualization of membrane protein topology

  • Live-cell single-molecule tracking using biotin-conjugated Fab fragments against TMEM43

• Single-cell analysis integration:

  • Coupling immunofluorescence using biotin-conjugated TMEM43 antibodies with single-cell RNA-seq

  • Mass cytometry (CyTOF) with metal-tagged streptavidin to quantify TMEM43 in heterogeneous cell populations

  • Spatial transcriptomics to correlate TMEM43 protein localization with local gene expression profiles

• Cryo-electron tomography:

  • Using biotin-conjugated TMEM43 antibodies with gold-streptavidin for precise localization in cellular ultrastructure

  • Correlative light and electron microscopy (CLEM) to bridge fluorescence imaging with ultrastructural analysis

How might biotin-conjugated TMEM43 antibodies contribute to understanding the role of this protein in auditory disorders?

Recent research has implicated TMEM43 mutations (p.R372X) in auditory disorders , presenting an emerging research area where biotin-conjugated antibodies could provide valuable insights:

Methodological approach for auditory research:

• Inner ear tissue analysis:

  • Optimize immunohistochemistry protocols for delicate cochlear structures

  • Use biotin-conjugated TMEM43 antibodies with streptavidin-fluorophores for high-sensitivity detection in low-abundance contexts

  • Implement clearing techniques (CLARITY, iDISCO) with streptavidin-compatible fluorophores for 3D visualization

• Developmental expression profiling:

  • Track TMEM43 expression during cochlear development using staged samples

  • Correlate expression with critical periods of auditory system maturation

  • Compare wild-type patterns with those in models expressing TMEM43 variants

• Functional studies in auditory cells:

  • Establish primary cultures of cochlear hair cells and supporting cells

  • Use biotin-conjugated TMEM43 antibodies to track protein dynamics during mechanotransduction

  • Implement patch-clamp electrophysiology with simultaneous immunofluorescence to correlate TMEM43 localization with channel function

• Translational research considerations:

  • Develop protocols for detecting TMEM43 in patient-derived samples

  • Establish biomarkers for auditory disorders linked to TMEM43 variants

  • Design screening approaches for TMEM43-targeted therapeutics

Research questions to address:

• Does TMEM43 function differ between cardiac and auditory tissues?

• What interaction partners are specific to TMEM43 in cochlear cells?

• How do different TMEM43 variants affect protein localization and function in auditory contexts?