MTERF4 Antibody, HRP conjugated

What is MTERF4 and why is it important in mitochondrial research?

MTERF4, or mitochondrial transcription termination factor 4, is a protein with a reported length of 381 amino acid residues and a mass of 44 kDa in humans. It is primarily localized in the mitochondria where it functions as a regulator of mitochondrial ribosome biogenesis and translation . This protein is also known by synonyms such as transcription termination factor 4 (mitochondrial) and MTERF domain-containing protein 2 (MTERFD2) . MTERF4 is important in mitochondrial research because it binds to mitochondrial ribosomal RNAs 16S, 12S, and 7S, and targets NSUN4 RNA methyltransferase to the mitochondrial large ribosomal subunit (39S) . Understanding MTERF4's role provides insights into mitochondrial translation processes and ribosomal assembly, which are critical for cellular energy production and homeostasis. The protein's conservation across various species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken suggests its fundamental importance in eukaryotic cellular function .

What are the common applications for MTERF4 antibodies in research?

MTERF4 antibodies are utilized across various experimental techniques in mitochondrial research. Western Blot represents the most common application for MTERF4 antibodies, allowing researchers to detect and quantify MTERF4 protein expression in cell or tissue lysates . Additionally, MTERF4 antibodies are frequently employed in Enzyme-Linked Immunosorbent Assay (ELISA) for quantitative detection of the protein in solution . Immunocytochemistry and Immunofluorescence techniques utilize these antibodies to visualize the subcellular localization of MTERF4 within cells, confirming its mitochondrial presence . Immunohistochemistry applications allow researchers to examine MTERF4 expression patterns in tissue sections, providing insights into tissue-specific distribution . The HRP-conjugated variants of MTERF4 antibodies are particularly valuable for ELISA applications due to their enhanced detection sensitivity through enzymatic signal amplification .

What are the differences between regular MTERF4 antibodies and HRP-conjugated versions?

Regular MTERF4 antibodies (unconjugated) require a secondary detection system, typically a species-specific secondary antibody that may itself be conjugated to an enzyme, fluorophore, or other detection tag. These unconjugated antibodies are versatile and can be used with different detection systems depending on the experimental requirements . In contrast, HRP-conjugated MTERF4 antibodies come with horseradish peroxidase directly attached to the antibody molecule . This direct conjugation eliminates the need for secondary antibodies, reducing the number of experimental steps and potentially decreasing background signal in assays like ELISA . The HRP enzyme catalyzes reactions that produce colorimetric, chemiluminescent, or fluorescent signals depending on the substrate used, enabling direct detection of the target protein . While unconjugated antibodies offer greater flexibility in detection methods, HRP-conjugated antibodies provide streamlined workflows, potentially higher sensitivity, and reduced cross-reactivity issues that can occur with secondary antibodies in complex samples.

How should MTERF4 antibodies, particularly HRP-conjugated versions, be stored and handled?

MTERF4 antibodies, including HRP-conjugated versions, require specific storage and handling conditions to maintain their functionality and reactivity. These antibodies should be stored at -20°C or -80°C for long-term preservation and stability . Repeated freeze-thaw cycles must be avoided as they can denature the antibody protein structure and reduce binding efficiency and enzymatic activity of the HRP conjugate . Most commercial MTERF4 antibodies are supplied in a buffer containing preservatives such as 0.03% Proclin 300 and stabilizers like 50% glycerol in PBS (pH 7.4) to maintain antibody integrity . When working with these antibodies, it is advisable to aliquot the stock solution into smaller volumes upon first thaw to minimize freeze-thaw cycles. For short-term storage during experiments, antibodies can be kept at 4°C for up to a week, but extended storage at this temperature may lead to gradual loss of activity, especially for the HRP component which is sensitive to temperature fluctuations and oxidation.

How does MTERF4's interaction with mitochondrial ribosomal RNAs affect experimental design?

MTERF4's binding to mitochondrial ribosomal RNAs (16S, 12S, and 7S) and its interaction with NSUN4 RNA methyltransferase represent critical aspects that must be considered in experimental design . When designing experiments to study MTERF4-ribosome interactions, researchers must account for the potential co-precipitation of these RNA species and associated proteins during immunoprecipitation assays . Cell fractionation protocols should be optimized to ensure proper isolation of mitoribosome complexes while maintaining the integrity of MTERF4's interactions with its binding partners . Crosslinking methods may be necessary to capture transient interactions between MTERF4 and mitochondrial RNAs or proteins like NSUN4 . Researchers should also consider that mutations or truncations in the MTERF domain could significantly alter RNA binding capabilities, potentially confounding results in overexpression or knockout studies . For RNA-protein interaction studies, techniques such as RNA immunoprecipitation (RIP) or crosslinking immunoprecipitation (CLIP) may be preferable to standard co-immunoprecipitation approaches to effectively capture the dynamic interactions between MTERF4 and its RNA binding partners within the mitochondrial environment.

What are the critical controls needed when using MTERF4 antibodies in mitochondrial research?

Implementing rigorous controls is essential when utilizing MTERF4 antibodies for mitochondrial research applications. Primary validation controls should include MTERF4 knockout or knockdown samples to confirm antibody specificity, as this eliminates the target protein and should result in absence of signal in assays like Western blot or immunostaining . Peptide competition assays represent another critical control wherein pre-incubation of the antibody with excess MTERF4 immunogen peptide (specifically the 45-87AA region) should block specific binding and diminish or eliminate specific signals . Cross-reactivity controls are particularly important given MTERF4's membership in the MTERF protein family, which contains structurally similar proteins that might be recognized by the antibody . Subcellular fractionation controls should include markers for various mitochondrial compartments (outer membrane, inner membrane, matrix) to confirm the expected mitochondrial localization of detected signals . For HRP-conjugated antibodies specifically, enzyme activity controls should be included to ensure that the conjugated HRP remains functional, particularly after storage or when using new lots of antibody . Additionally, tissue-specific expression patterns should be verified across multiple species when conducting comparative studies, as MTERF4 orthologs have been reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken models .

How do MTERF4's interactions with mitoribosomes affect antibody selection for complex analysis?

MTERF4's strong interaction with mitoribosomes and its role in targeting NSUN4 RNA methyltransferase to the large ribosomal subunit necessitates careful antibody selection for complex analyses . When studying MTERF4 within mitoribosomal complexes, researchers should select antibodies that target epitopes known to remain accessible in the assembled complex, avoiding regions involved in protein-protein or protein-RNA interactions . For co-immunoprecipitation studies of MTERF4-mitoribosome interactions, antibodies recognizing the N-terminal or C-terminal regions may be preferable if these domains extend outward from the complex . Researchers must consider that certain fixation methods used in immunohistochemistry or immunofluorescence might disrupt or alter the conformation of MTERF4-mitoribosome complexes, potentially masking epitopes or creating artifacts . For studies examining dynamic assembly of mitoribosomes, non-denaturing conditions and native-PAGE approaches combined with antibodies validated under native conditions would be optimal to preserve complex integrity . The HRP-conjugated versions of MTERF4 antibodies may have limited utility in certain complex analyses due to the potential steric hindrance from the HRP moiety, which might interfere with binding to MTERF4 when it is engaged in large macromolecular assemblies with mitoribosomal components .

What optimization steps are necessary for Western Blot applications using MTERF4 antibodies?

Optimizing Western Blot protocols for MTERF4 detection requires several critical adjustments to achieve specific and sensitive results. Mitochondrial enrichment through subcellular fractionation is often necessary before sample preparation due to MTERF4's mitochondrial localization, which increases signal-to-noise ratio compared to whole cell lysates . Sample preparation conditions must be carefully controlled, as MTERF4's association with mitoribosomal complexes may require more stringent lysis buffers containing ionic detergents like SDS to fully solubilize and denature these complexes . Blocking conditions should be empirically determined, with 5% non-fat dry milk in TBST generally serving as a starting point, though BSA-based blocking may be preferable when using phospho-specific antibodies targeting MTERF4 post-translational modifications . When working with HRP-conjugated MTERF4 antibodies, the antibody dilution must be carefully optimized (typically starting at 1:1000 to 1:5000) to balance specific signal detection with minimal background . Extended primary antibody incubation times (overnight at 4°C) often yield better results than shorter incubations at room temperature due to the typically low abundance of MTERF4 in many cell types . For chemiluminescent detection, enhanced chemiluminescence (ECL) substrates with longer signal duration are recommended to allow for multiple exposures when determining the optimal signal intensity range without saturation.

How should ELISA protocols be modified when using HRP-conjugated MTERF4 antibodies?

ELISA protocols using HRP-conjugated MTERF4 antibodies require specific modifications to maximize sensitivity and specificity. The direct detection approach possible with HRP-conjugated antibodies eliminates the secondary antibody incubation step, significantly reducing the total assay time while potentially improving signal-to-noise ratios . Antibody concentration optimization is particularly critical with HRP-conjugated antibodies, as both under and over-concentration can lead to suboptimal results; titration experiments starting from 1:500 to 1:5000 dilutions should be performed to determine the optimal concentration for each specific application and sample type . Blocking buffers may need adjustment when working with HRP-conjugated antibodies, with protein-free blockers sometimes providing advantages by eliminating potential cross-reactivity with the conjugated enzyme . Wash steps following antibody incubation must be thorough (typically 4-5 washes) to remove unbound HRP-conjugated antibody and reduce background signal, using PBS or TBS with 0.05-0.1% Tween-20 . Substrate selection is crucial for HRP-conjugated antibodies, with TMB (3,3',5,5'-tetramethylbenzidine) often preferred for its sensitivity, though other options like ABTS or OPD may be suitable depending on the specific requirements for detection range and signal stability . Signal development time must be carefully monitored and optimized for HRP-conjugated antibodies to avoid substrate depletion or excessive background development, typically requiring kinetic readings or timed endpoint measurements.

What factors affect the performance of MTERF4 antibodies in immunofluorescence applications?

Multiple factors significantly impact the performance of MTERF4 antibodies in immunofluorescence applications, requiring careful consideration during experimental design. Fixation methods dramatically influence epitope accessibility and antibody binding, with paraformaldehyde fixation (typically 4%) for 10-15 minutes representing a standard starting point, though methanol fixation may better preserve certain MTERF4 epitopes while simultaneously permeabilizing mitochondrial membranes . Permeabilization conditions must be optimized specifically for mitochondrial targets like MTERF4, with Triton X-100 (0.1-0.2%) or digitonin (10-50 μg/ml) being common choices that provide different degrees of membrane disruption while maintaining mitochondrial structure . Mitochondrial counterstaining with markers such as MitoTracker or antibodies against established mitochondrial proteins (COX IV, TOM20) should be employed to confirm the expected subcellular localization of MTERF4 signals and rule out non-specific binding to other cellular compartments . The use of super-resolution microscopy techniques such as STED (Stimulated Emission Depletion) or STORM (Stochastic Optical Reconstruction Microscopy) may be necessary to precisely localize MTERF4 within mitochondrial subcompartments due to the organelle's small size and the specific association of MTERF4 with mitoribosomes . For HRP-conjugated MTERF4 antibodies, which are not typically used in immunofluorescence, conversion to fluorescent signals can be achieved through tyramide signal amplification (TSA) methods, though this adds complexity and potential sources of background to the protocol .

How can researchers address common issues with MTERF4 antibody specificity?

Addressing specificity issues with MTERF4 antibodies requires systematic troubleshooting approaches and validation strategies. Epitope mapping represents a critical first step when specificity problems arise, as understanding exactly which region of MTERF4 the antibody recognizes (such as the 45-87AA region used as immunogen for some commercial antibodies) helps identify potential cross-reactivity with related proteins containing similar epitopes . Pre-adsorption tests with recombinant MTERF4 or the specific immunogen peptide should eliminate specific signals while leaving non-specific signals unchanged, providing clear evidence of antibody specificity . Cross-reactivity with other MTERF family members (MTERF1, MTERF2, MTERF3) represents a common specificity issue that can be evaluated by testing the antibody against recombinant versions of these related proteins or in knockout/knockdown systems for each family member . Western blot analysis should confirm a single band at the expected molecular weight of 44 kDa for human MTERF4, though post-translational modifications may result in slight mobility shifts that should be characterized . Genetic validation using CRISPR/Cas9-mediated knockout or RNAi-mediated knockdown of MTERF4 provides the most definitive specificity control, as these interventions should abolish or significantly reduce specific antibody signals across all applications . Comparative analysis across multiple antibodies targeting different epitopes of MTERF4 can provide additional confidence in specificity when consistent localization or expression patterns are observed.

What are the best practices for quantifying MTERF4 levels using antibody-based methods?

Accurate quantification of MTERF4 levels requires rigorous methodological approaches and appropriate normalization strategies. Standard curve generation using recombinant MTERF4 protein at known concentrations is essential for absolute quantification in ELISA assays, with serial dilutions covering the expected physiological range (typically 0.1-100 ng/ml) and analyzed using four-parameter logistic regression . Sample preparation standardization is critical, as variations in extraction efficiency from mitochondria can significantly impact measured MTERF4 levels; consistent protocols for mitochondrial isolation and protein extraction should be established and strictly followed across all experimental conditions . Normalization strategies should include both total protein normalization (BCA or Bradford assays) and specific mitochondrial markers (such as TOM20, VDAC, or citrate synthase activity) to account for variations in mitochondrial content between samples . For Western blot quantification, densitometric analysis should be performed within the linear dynamic range of detection, which requires preliminary experiments with serial dilutions of samples to establish appropriate loading amounts and exposure times . Biological replicates (minimum n=3) and technical replicates are essential for establishing statistical significance of observed differences in MTERF4 levels, with appropriate statistical tests selected based on data distribution and experimental design . Batch effects should be minimized by processing experimental and control samples simultaneously, and when this is not possible, including common reference samples across batches to allow for inter-batch normalization.

How can researchers reliably interpret results from co-localization studies involving MTERF4?

Reliable interpretation of co-localization studies involving MTERF4 requires sophisticated imaging approaches and quantitative analysis. Confocal microscopy with appropriate resolution settings (optimally approaching the diffraction limit of approximately 200 nm laterally and 500 nm axially) represents the minimum imaging requirement for mitochondrial co-localization studies, given the organelle's small size and complex internal structure . Z-stack acquisition and 3D reconstruction should be employed to fully capture the three-dimensional distribution of MTERF4 and potential co-localization partners within mitochondria, avoiding artifacts from single optical sections . Quantitative co-localization analysis should utilize established metrics such as Pearson's correlation coefficient, Manders' overlap coefficient, or object-based co-localization methods rather than relying solely on visual assessment of color overlap in merged images . Super-resolution techniques including STED, STORM, or PALM may be necessary to accurately resolve MTERF4's association with mitoribosomes and other mitochondrial components, providing spatial resolution down to 20-50 nm . Proximity ligation assays (PLA) offer an alternative approach for verifying protein-protein interactions involving MTERF4 within a range of approximately 40 nm, providing greater specificity than standard co-localization methods for confirming direct molecular associations . Live-cell imaging approaches using appropriate fluorescent protein fusions can complement fixed-cell studies by revealing the dynamics of MTERF4 interactions, though careful validation is needed to ensure that such fusions do not disrupt MTERF4's normal function or localization .