MTERF4 Antibody, Biotin conjugated

What is MTERF4 and what cellular functions does it serve?

MTERF4 (Mitochondrial Transcription Termination Factor 4) functions as a regulator of mitochondrial ribosome biogenesis and translation. It binds to mitochondrial ribosomal RNAs, including 16S, 12S, and 7S, and plays a crucial role in targeting NSUN4 RNA methyltransferase to the mitochondrial large ribosomal subunit (39S) . Unlike other members of the mTERF family that may function in both organelles, MTERF4 appears to be primarily localized to chloroplasts rather than mitochondria in plants, as shown in studies with the maize ortholog Zm-mTERF4 . The functional role of MTERF4 has been further characterized in ribosome assembly, where it forms a heterodimer with NSUN4 that is critical for mitoribosome maturation .

How does MTERF4 differ from other mTERF family proteins?

While all mTERF proteins share structural similarities in their mTERF domains, they fulfill distinct cellular functions. MTERF4 specifically functions in ribosome biogenesis, whereas other family members like mTERF8 are involved in transcription termination of specific genes such as the chloroplast gene psbJ . MTERF4 forms a specific functional heterodimer with NSUN4 that plays a role in exposing the peptidyl transferase region during human mitoribosomal assembly . Unlike some mTERF proteins that may have dual targeting to both mitochondria and chloroplasts, localization studies of Zm-mTERF4 (maize ortholog) suggest it is predominantly found in chloroplasts and absent from mitochondria .

What is the structural basis for MTERF4's function in ribosome assembly?

MTERF4 protein folds into a bent α-solenoid structure that binds with its convex region to the surface of the immature ribosomal subunit. Its positively charged concave region is exposed toward the outside where it binds double-helical RNA . Through these interactions, MTERF4 exposes immature rRNA regions corresponding to the peptidyl transferase (PTC) loop that forms the active site of the ribosome. This structural arrangement is crucial for the stepwise maturation of functionally important regions in the human mitochondrial large ribosomal subunit .

What experimental techniques can be combined with MTERF4 antibodies to study RNA-protein interactions?

For studying RNA-protein interactions involving MTERF4, researchers can employ RNA coimmunoprecipitation assays (RIP-chip) using affinity-purified anti-MTERF4 antibodies. This technique can be performed with stromal protein extract and analyzed using various array formats, including custom high-resolution microarrays with synthetic oligonucleotides tiling all annotated transcripts of interest . Additionally, researchers studying MTERF family proteins have successfully employed Electrophoretic Mobility Shift Assays (EMSA) to demonstrate specific binding to target DNA sequences, as exemplified with mTERF8 . ChIP analysis with appropriate antibodies can also be used to confirm binding sites, as demonstrated in complemented plants expressing tagged versions of mTERF proteins .

How should immunoprecipitation experiments with MTERF4 antibodies be designed and optimized?

Immunoprecipitation experiments with MTERF4 antibodies should include careful selection of extraction conditions to maintain protein-RNA or protein-protein interactions. Based on protocols used for related studies, stromal protein extract preparation should be optimized according to established methods . When designing such experiments, include appropriate negative controls such as immunoprecipitation with antibodies against unrelated proteins (e.g., OE16 or AtpB as used in published studies) . For validation of results, consider performing replicate experiments using different array designs and independent protein preparations. Quantitative analysis of immunoprecipitated material can be performed using techniques such as RT-qPCR for bound nucleic acids .

What cellular fractionation approaches are appropriate when working with MTERF4 antibodies?

Based on research with Zm-mTERF4, cellular fractionation approaches should include isolation of organelles (chloroplasts and mitochondria) followed by subfractionation of chloroplasts to separate stromal, thylakoid, and membrane fractions . When studying MTERF4 in chloroplasts, the stroma fraction should be carefully isolated as Zm-mTERF4 has been found primarily in this fraction. Validation of fractionation quality should include immunoblotting with markers for different cellular compartments to ensure purity of fractions. For analysis of protein complexes containing MTERF4, consider blue native (BN) gel electrophoresis followed by SDS-PAGE in the second dimension, as this approach has been successful for related mTERF proteins .

What are common issues in Western blot detection using biotin-conjugated MTERF4 antibodies and how can they be addressed?

When performing Western blots with biotin-conjugated MTERF4 antibodies, researchers may encounter high background due to endogenous biotin-containing proteins. To address this, pre-block membranes with avidin or streptavidin followed by biotin blocking solution before applying the biotin-conjugated antibody. Signal detection should utilize streptavidin-conjugated reporter systems rather than secondary antibodies. Based on the molecular weight of MTERF4 (approximately 49 kDa), optimization of gel percentage and transfer conditions may be necessary to achieve clear detection . Additionally, as observed with Zm-mTERF4 detection, protein abundance may vary with tissue developmental stage, so sampling from appropriate tissues is crucial for detection of adequate protein levels .

How can specificity of MTERF4 antibody be validated for research applications?

Validation of MTERF4 antibody specificity should include multiple approaches. First, comparison of protein detection between wild-type samples and those with reduced MTERF4 expression (e.g., mutants or knockdown lines) can confirm specificity, as demonstrated with Zm-mTERF4 where antibody detected a protein of expected size that was reduced in mutant lines . Second, recombinant MTERF4 protein can be used as a positive control. Third, immunoprecipitation followed by mass spectrometry can verify that the antibody is capturing the intended target. For biotin-conjugated antibodies, additional controls should test for potential cross-reactivity with biotin-binding proteins independently of the primary antibody specificity .

What are the critical considerations for proper storage and handling of biotin-conjugated MTERF4 antibodies?

Biotin-conjugated MTERF4 antibodies require specific storage and handling conditions to maintain functionality. According to product specifications, the antibody should be shipped at 4°C and upon delivery aliquoted and stored at -20°C or -80°C . Repeated freeze-thaw cycles should be avoided to prevent degradation of the antibody and loss of biotin conjugation efficiency. The storage buffer containing 50% glycerol and 0.03% Proclin 300 as a preservative helps maintain stability . When preparing working dilutions, use buffers matching the storage buffer's pH (7.4) and include appropriate stabilizing proteins. For long-term studies, monitor the performance of the antibody over time by including positive controls in each experiment to detect any reduction in binding efficiency.

How can MTERF4 antibodies be utilized to investigate protein-complex formation during ribosome assembly?

MTERF4 antibodies can be employed in co-immunoprecipitation studies to capture and analyze the composition of protein complexes during ribosome assembly. Based on structural studies, MTERF4 forms a heterodimer with NSUN4 that binds to immature mitoribosomal subunits . Researchers can use biotin-conjugated MTERF4 antibodies with streptavidin beads to pull down these complexes, followed by mass spectrometry to identify associated proteins. To investigate the dynamics of complex formation, consider performing these experiments across a time course of ribosome assembly. Complementary approaches such as blue native gel electrophoresis followed by immunoblotting can reveal the size and composition of native complexes containing MTERF4, similar to methods used for mTERF8 . When interpreting results, account for the observed structural relationships between MTERF4 and rRNA regions corresponding to the peptidyl transferase active site cleft .

What approaches can be used to study the interaction between MTERF4 and NSUN4 in different cellular contexts?

To study the MTERF4-NSUN4 interaction, researchers can employ multiple complementary approaches. In vitro binding assays using recombinant proteins can establish direct interaction, while co-immunoprecipitation with MTERF4 antibodies can confirm the interaction in cellular contexts. Proximity ligation assays offer another approach to visualize interactions in situ. Based on structural studies, the MTERF4-NSUN4 heterodimer functions in mitoribosome maturation through interactions with specific rRNA regions . To understand the functional significance of this interaction in different cellular contexts, consider comparative studies in various tissues or under conditions that affect mitochondrial translation. Mutations in the interaction interface between these proteins can be introduced to assess the impact on complex formation and downstream functions in ribosome assembly .

How can researchers employ MTERF4 antibodies to investigate the relationship between mitochondrial ribosome assembly defects and disease states?

MTERF4 antibodies can be valuable tools for investigating mitochondrial ribosome assembly defects in disease contexts. Researchers can compare MTERF4 expression, localization, and complex formation between normal and disease-state tissues or cell lines using immunoblotting, immunofluorescence, or immunoprecipitation approaches. Given MTERF4's role in mitoribosome assembly and specifically in maturation of the peptidyl transferase region , analysis of translation efficiency in correlation with MTERF4 function could provide insights into disease mechanisms. Biotin-conjugated antibodies offer advantages for multiplexed detection approaches, allowing simultaneous visualization of MTERF4 with other markers of mitochondrial function. For clinical samples with limited material, consider using highly sensitive detection methods with the biotin-conjugated antibody to maximize signal from minimal input material .

How should researchers interpret differences in MTERF4 detection between tissue types or developmental stages?

When interpreting differences in MTERF4 detection across tissue types or developmental stages, researchers should consider multiple factors. Studies with Zm-mTERF4 revealed that protein abundance increases along developmental gradients in maize leaves, with lower levels in basal (younger) sections and higher levels in apical (more mature) sections . This pattern suggests that MTERF4 accumulation correlates with organelle maturation. When quantifying such differences, normalization to appropriate loading controls is essential. Additionally, researchers should distinguish between changes in protein expression versus protein stability; in hypomorphic mutants, reduced synthesis rates were partially compensated by increased protein stability as chloroplast development proceeded . Consider complementing protein detection with transcript analysis to determine whether differences originate at transcriptional or post-transcriptional levels.

What considerations are important when analyzing MTERF4 binding to nucleic acids through antibody-based approaches?

Analysis of MTERF4 binding to nucleic acids requires careful consideration of experimental design and controls. Based on studies of mTERF family proteins, specific binding should be confirmed using competition assays with unlabeled probes, and specificity should be validated using unrelated nucleic acid sequences as negative controls . When interpreting RIP-chip or similar data, factor in the possibility of indirect interactions, as MTERF4 may bind as part of larger complexes. Statistical analysis should include normalization to input material and comparison to negative control immunoprecipitations with unrelated antibodies . For biotin-conjugated antibodies specifically, ensure that streptavidin-based detection systems don't introduce artifacts from endogenous biotinylated proteins. When mapping binding sites, use overlapping probes or fragments to precisely define interaction regions, as demonstrated in studies with mTERF8 .

How can researchers differentiate between direct and indirect effects when studying MTERF4 function using antibody-based approaches?

Differentiating between direct and indirect effects in MTERF4 functional studies requires careful experimental design. For binding studies, compare in vitro binding of recombinant MTERF4 to potential targets versus binding observed in cellular contexts. Based on structural studies, MTERF4 directly interacts with specific rRNA regions and forms a heterodimer with NSUN4 , but may indirectly affect other processes. To distinguish primary from secondary effects, conduct time-course experiments to establish the sequence of events following perturbation of MTERF4 function. Additionally, use targeted mutations that disrupt specific interactions rather than complete protein depletion. When interpreting ribosome assembly defects, consider that MTERF4 specifically affects maturation of the peptidyl transferase region , so defects in this process likely represent direct effects, while broader translation defects may be secondary consequences.