TMEM243 Antibody

What is TMEM243 and why is it significant in research?

TMEM243, also known as Transmembrane Protein 243 Mitochondrial, is a mitochondrial membrane protein previously identified as C7orf23 (Chromosome 7 Open Reading Frame 23). It has also been referred to as MDR1- and mitochondrial taxol resistance-associated protein (MM-TRAG), suggesting potential roles in drug resistance mechanisms . TMEM243 is significant in research due to its mitochondrial localization and possible implications in cellular processes related to mitochondrial function, though its precise biological functions remain under investigation.

What applications are TMEM243 antibodies suitable for?

TMEM243 antibodies have been validated for several key research applications. Based on available product information, these antibodies are suitable for ELISA (Enzyme-Linked Immunosorbent Assay), Immunohistochemistry (IHC), and Immunofluorescence (IF) techniques . These applications enable researchers to detect and visualize TMEM243 protein in various experimental contexts, from protein quantification to cellular localization studies.

What is the recommended dilution range for TMEM243 antibodies in different applications?

For optimal results across different applications, TMEM243 antibodies should be used at specific dilution ranges. For immunohistochemistry (IHC), the recommended dilution range is 1:20-1:200, while for immunofluorescence (IF), the optimal range is 1:50-1:200 . These ranges should be considered starting points, and researchers may need to perform titration experiments to determine the optimal concentration for their specific experimental conditions and sample types.

What are the proper storage conditions for TMEM243 antibodies?

TMEM243 antibodies require specific storage conditions to maintain their activity and specificity. The recommended storage temperature is -20°C to -80°C . Upon receipt, antibodies should be stored at these temperatures to preserve functionality. It's important to note that repeated freeze-thaw cycles should be avoided as they can compromise antibody quality. When working with the antibody, aliquoting into single-use volumes is advisable to minimize freeze-thaw cycles .

How can specificity of TMEM243 antibody be validated in experimental systems?

Validating the specificity of TMEM243 antibody requires a multi-faceted approach. First, perform western blotting using both recombinant TMEM243 protein and cell lysates expressing TMEM243, looking for a single band at the expected molecular weight of approximately 13.2 kDa . Second, conduct immunoprecipitation followed by mass spectrometry to confirm the identity of the precipitated protein. Third, utilize TMEM243 knockout or knockdown cell lines as negative controls to confirm signal specificity. Finally, peptide competition assays can be performed using the specific immunogen (amino acids 1-31 of human TMEM243) to further demonstrate binding specificity.

What considerations should be taken into account when using TMEM243 antibody for co-localization studies with mitochondrial markers?

When conducting co-localization studies of TMEM243 with mitochondrial markers, several methodological considerations are critical. First, select appropriate mitochondrial markers that don't interfere with TMEM243 detection (e.g., MitoTracker dyes or antibodies against distinct mitochondrial proteins such as TOM20 or COXIV). Second, optimize fixation protocols—paraformaldehyde (4%) generally preserves mitochondrial morphology while maintaining antigen accessibility. Third, implement proper controls including single-staining controls to assess bleed-through and co-localization with other organelle markers to confirm specificity. Finally, use high-resolution imaging techniques such as confocal or super-resolution microscopy for accurate co-localization analysis, followed by quantitative assessment using coefficients such as Pearson's or Mander's.

How should researchers approach TMEM243 antibody validation in immunohistochemistry applications?

Validating TMEM243 antibody for immunohistochemistry requires systematic optimization and controls. Begin with antigen retrieval optimization, testing both heat-induced epitope retrieval (HIER) methods with citrate (pH 6.0) and EDTA (pH 9.0) buffers. Next, perform a dilution series (1:20 to 1:200) on positive control tissues known to express TMEM243 . Include negative controls consisting of isotype-matched antibodies and tissues known not to express TMEM243. Furthermore, verify staining patterns by comparing with data from orthogonal methods such as in situ hybridization or RNA-seq tissue expression data. Finally, if available, validate results using tissues from TMEM243 knockout models or tissues treated with TMEM243-specific siRNA.

What are the critical considerations when designing experiments to investigate TMEM243's role in mitochondrial function?

Investigating TMEM243's role in mitochondrial function requires a comprehensive experimental approach. First, establish TMEM243 knockdown or knockout models using siRNA, shRNA, or CRISPR-Cas9 systems, coupled with appropriate rescue experiments using wild-type TMEM243 expression constructs. Second, assess key mitochondrial parameters including membrane potential (using JC-1 or TMRM dyes), respiratory capacity (using Seahorse XF analyzers), ROS production (using MitoSOX), and mitochondrial morphology (using electron microscopy or super-resolution fluorescence microscopy). Third, examine mitochondrial protein import mechanisms, as transmembrane proteins often participate in these processes. Finally, investigate potential protein-protein interactions within the mitochondrial membrane using techniques such as proximity labeling (BioID) or co-immunoprecipitation followed by mass spectrometry.

What controls should be included when using TMEM243 antibody in immunofluorescence studies?

When conducting immunofluorescence experiments with TMEM243 antibody, a comprehensive set of controls is essential. Include primary antibody controls (using the same dilution of isotype-matched antibody from the same species) to identify non-specific binding. Implement secondary antibody-only controls to detect background fluorescence. Use positive controls consisting of cells or tissues known to express TMEM243. Include negative controls using cells with TMEM243 knockdown/knockout or tissues known not to express the protein. For co-localization studies, prepare single-label controls to assess spectral bleed-through. Finally, perform peptide competition assays using the immunizing peptide (AA 1-31 of human TMEM243) to confirm antibody specificity.

How can researchers troubleshoot weak or absent signals when using TMEM243 antibody?

When encountering weak or absent signals with TMEM243 antibody, follow this systematic troubleshooting approach. First, verify antibody viability through dot blot analysis with the immunizing peptide. Next, optimize antibody concentration by testing broader dilution ranges than the recommended 1:20-1:200 for IHC or 1:50-1:200 for IF . Enhance signal detection by implementing amplification systems such as tyramide signal amplification or more sensitive detection systems. Optimize fixation conditions, testing both cross-linking (paraformaldehyde) and precipitating (methanol/acetone) fixatives. Improve antigen retrieval by testing different buffer compositions and incubation times. Finally, evaluate sample expression levels through RT-qPCR to confirm TMEM243 expression in your experimental system, as expression levels may vary across cell types and tissues.

What are the key considerations for using TMEM243 antibody in co-immunoprecipitation experiments?

When using TMEM243 antibody for co-immunoprecipitation, several critical factors must be addressed. Begin by selecting appropriate lysis conditions that preserve protein interactions—mild non-ionic detergents (0.5-1% NP-40 or Triton X-100) are generally suitable for membrane proteins like TMEM243. Pre-clear lysates thoroughly to reduce non-specific binding. Consider cross-linking approaches (such as DSP or formaldehyde) to stabilize transient interactions. Use sufficient antibody amounts (typically 2-5 μg per mg of protein lysate) and optimize incubation conditions (4°C overnight on a rotator). Select appropriate beads (Protein G for rabbit polyclonal antibodies) and include stringent washing steps with decreasing detergent concentrations. Implement proper controls including IgG control, input samples, and ideally, immunoprecipitation from TMEM243-depleted samples as negative controls.

What methodological approach should be used to investigate potential interaction partners of TMEM243?

Investigating TMEM243 interaction partners requires a multi-technique approach. First, perform standard co-immunoprecipitation using TMEM243 antibody followed by mass spectrometry to identify potential binding partners. Complement this with proximity-dependent approaches such as BioID or APEX2, where TMEM243 is fused to a biotin ligase to label proximal proteins. Implement FRET or BRET analysis for investigating specific protein pair interactions in living cells. Conduct yeast two-hybrid screening as an orthogonal approach to identify direct protein-protein interactions. Verify key interactions through reciprocal co-immunoprecipitation, co-localization studies, and functional assays. Consider examining interaction dynamics under different cellular conditions (e.g., stress, differentiation) to identify context-dependent interactions.

How should researchers interpret cross-reactivity data for TMEM243 antibody?

Interpreting cross-reactivity data for TMEM243 antibody requires careful consideration of several factors. According to available information, the antibody shows reactivity with human TMEM243 . When evaluating potential cross-reactivity, analyze sequence homology of the immunizing peptide (AA 1-31) across species and related proteins. Validate cross-reactivity experimentally using western blotting with lysates from multiple species and observe if bands appear at the expected molecular weight (approximately 13.2 kDa) . When unexpected bands appear, perform peptide competition assays to determine if they represent specific or non-specific binding. Consider cross-reactivity data in the context of your experimental system, and if working with non-human systems, validate the antibody specifically in your species of interest through orthogonal approaches.

What approaches can be used to quantify TMEM243 expression levels in different experimental conditions?

Quantifying TMEM243 expression levels across experimental conditions requires a multi-method approach. For protein-level quantification, implement western blotting with careful normalization to appropriate loading controls (preferably multiple controls including a mitochondrial protein and a general housekeeping protein). Use ELISA assays with recombinant TMEM243 protein as standards for absolute quantification. For tissue or cellular distribution analysis, apply quantitative immunofluorescence or immunohistochemistry with automated image analysis software for objective quantification. At the mRNA level, use RT-qPCR with validated primers and appropriate reference genes. For broader studies, implement targeted proteomics approaches using selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) with isotopically labeled peptide standards for absolute quantification of TMEM243 protein.

How can researchers differentiate between specific and non-specific binding in TMEM243 antibody applications?

Differentiating between specific and non-specific binding requires rigorous control experiments and analytical approaches. First, perform peptide competition assays using the immunizing peptide (AA 1-31) —specific signals should be substantially reduced. Second, compare staining/binding patterns across multiple TMEM243 antibodies targeting different epitopes—concordant patterns suggest specificity. Third, correlate protein detection with mRNA expression data from RT-qPCR or public databases. Fourth, implement genetic approaches by comparing signal between wild-type samples and those with TMEM243 knockdown/knockout—specific signals should be reduced or eliminated in knockout samples. Finally, for immunofluorescence applications, evaluate subcellular localization patterns—TMEM243 signals should predominately co-localize with mitochondrial markers given its mitochondrial localization.

How should researchers approach epitope mapping of TMEM243 antibody?

Epitope mapping of TMEM243 antibody requires a systematic approach combining computational and experimental methods. Begin with computational prediction based on the known immunizing sequence (AA 1-31) to identify potential antibody binding sites. Experimentally, perform peptide array analysis using overlapping peptides spanning the immunizing region to pinpoint the exact binding epitope. Conduct alanine scanning mutagenesis, creating point mutations throughout the epitope region and testing antibody binding to identify critical residues. Implement hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions protected from exchange upon antibody binding. For structural characterization, consider X-ray crystallography or cryo-EM of the antibody-epitope complex. Finally, conduct cross-reactivity analysis with related proteins containing similar sequences to assess epitope uniqueness.

What methodological considerations are important when developing a quantitative ELISA for TMEM243?

Developing a quantitative ELISA for TMEM243 requires careful optimization of multiple parameters. First, select an appropriate format—sandwich ELISA is preferred for complex samples, requiring two antibodies recognizing different epitopes, while direct ELISA may be suitable for purified samples. Prepare high-quality standards using recombinant TMEM243 protein with verified purity (>80% as determined by SDS-PAGE) . Optimize coating conditions (concentration, buffer, time) and blocking parameters to minimize background. Determine optimal antibody concentrations through checkerboard titration, starting with the recommended dilutions for ELISA applications . Validate assay performance by assessing linearity, recovery, precision, and limits of detection/quantification across multiple runs. Implement spike-recovery experiments to evaluate matrix effects in complex samples. Finally, establish appropriate quality control measures including positive and negative controls for each assay run.

Table 1: Optimized Dilution Ranges for TMEM243 Antibody Applications

Data compiled from product information . RT = room temperature.

Table 2: Technical Specifications of TMEM243 Antibody and Protein

Data compiled from product specifications .

How might TMEM243 antibodies be used to investigate potential roles in drug resistance mechanisms?

Given TMEM243's alternative name as MDR1- and mitochondrial taxol resistance-associated protein (MM-TRAG) , investigating its role in drug resistance requires specific methodological approaches. Develop cellular models with TMEM243 overexpression, knockdown, and knockout to evaluate changes in chemotherapy sensitivity, particularly to taxol compounds. Implement time-course studies using TMEM243 antibodies to monitor protein expression changes before and after drug exposure. Perform co-localization studies with drug transporters like MDR1/P-glycoprotein to assess potential functional interactions. Utilize proximity labeling techniques (BioID/APEX) with TMEM243 as bait to identify interacting proteins involved in drug response pathways. Conduct subcellular fractionation followed by western blotting to monitor TMEM243 redistribution during drug treatment. Finally, correlate TMEM243 expression levels with drug response metrics in clinical samples using IHC with validated cutoff scores.

What research strategies would be most effective for elucidating TMEM243's function in mitochondrial biology?

Elucidating TMEM243's mitochondrial function requires a comprehensive research strategy. First, generate fluorescent protein-tagged TMEM243 constructs to precisely visualize its submitochondrial localization through super-resolution microscopy. Conduct mitochondrial subfractionation experiments followed by western blotting to biochemically confirm its localization to specific mitochondrial compartments (outer membrane, inner membrane, matrix, or intermembrane space). Implement CRISPR-Cas9 gene editing to create TMEM243 knockout cell lines for comprehensive phenotypic analysis. Assess effects on mitochondrial functions including respiration (Seahorse assays), membrane potential (TMRM staining), morphology (electron microscopy), and dynamics (fission/fusion events). Apply quantitative proteomics to identify changes in the mitochondrial proteome following TMEM243 manipulation. Finally, investigate potential roles in specific mitochondrial processes such as protein import, lipid metabolism, or apoptosis regulation through targeted functional assays.