MRPL45 Antibody

What is MRPL45 and what is its biological function?

MRPL45 (Mitochondrial Ribosomal Protein L45) is a component of the mitochondrial large ribosomal subunit (mt-LSU). The protein plays a crucial role in mitochondrial translation by directing the nascent polypeptide toward the tunnel exit and positioning the exit at a distance from the membrane surface . This 35 kDa protein interacts directly with the inner mitochondrial membrane (IMM), potentially serving as an anchor point between the mitoribosome and the membrane .

Functionally, MRPL45 is essential for the stability of the mt-LSU and proper mitochondrial translation. Depletion studies have confirmed that reduced MRPL45 leads to instability of the mitoribosomal large subunit and compromised mitochondrial protein synthesis . Decreased or malfunctioning MRPL45 is associated with mitochondrial disorders, which often manifest as varied clinical symptoms due to impaired energy production .

What sample types and applications have been validated for MRPL45 antibodies?

MRPL45 antibodies have been validated across multiple applications and sample types as summarized in the following table:

These antibodies have demonstrated reactivity with human, mouse, and rat samples . For IHC applications, antigen retrieval with TE buffer pH 9.0 is suggested, although citrate buffer pH 6.0 may serve as an alternative . Over 13 publications have utilized these antibodies for Western blot applications, and 2 publications have employed them for immunofluorescence studies .

What are the optimal storage conditions for MRPL45 antibodies?

MRPL45 antibodies should be stored at -20°C, where they remain stable for one year after shipment . The antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Importantly, aliquoting is unnecessary for -20°C storage, simplifying laboratory handling procedures. Some antibody preparations (20μl sizes) may contain 0.1% BSA as a stabilizing agent .

For long-term preservation of antibody activity, avoid repeated freeze-thaw cycles and contamination. When working with the antibody, briefly centrifuge the vial before opening to ensure collection of all liquid at the bottom of the tube.

What controls should be included when using MRPL45 antibodies?

When using MRPL45 antibodies, several controls should be implemented to ensure experimental validity:

• Positive controls: Use cell lines with confirmed MRPL45 expression such as HeLa or SKOV-3 cells for validating antibody performance .

• Negative controls: Include samples where primary antibody is omitted or replaced with non-specific IgG to assess background staining.

• Knockdown/knockout validation: When available, samples with MRPL45 depletion provide excellent specificity controls. Studies have shown that siRNA-mediated depletion of MRPL45 confirms antibody specificity and also demonstrates the protein's importance for mt-LSU stability .

• Loading controls: For Western blot applications, appropriate loading controls (mitochondrial for fractionated samples, or housekeeping proteins for whole cell lysates) should be included.

• Cross-reactivity assessment: When working with multi-species samples, evaluate potential cross-reactivity by testing the antibody against purified recombinant proteins or well-characterized cell lines from different species.

How can MRPL45 antibodies be used to investigate mitoribosome assembly defects?

MRPL45 antibodies provide valuable tools for investigating mitoribosome assembly defects, particularly in the context of disease. Research approaches include:

• Quantitative proteomics coupled with immunoprecipitation: MRPL45 antibodies can be used to immunoprecipitate the protein along with its interacting partners for mass spectrometry analysis. This approach helps identify components of subcomplexes formed during mt-LSU biogenesis . Recent multi-omics studies have employed quantitative proteomics to detect protein signatures characteristic of mitoribosomal defects, showing that MRPL45 and other MRPLs exhibit specific fold change patterns in patients with mitochondrial disorders .

• Relative Complex Abundance analysis: This proteomics-based method can identify defects in oxidative phosphorylation (OXPHOS) disorders with high sensitivity. In patients with mitoribosomal gene mutations (such as in MRPL39 and MRPL15), specific patterns of mitoribosomal protein reduction can be observed, with MRPL45 serving as an indicator of mt-LSU stability .

• Gradient fractionation with Western blot: By combining sucrose gradient fractionation with Western blot analysis using MRPL45 antibodies, researchers can track the incorporation of MRPL45 into the mt-LSU during assembly. This technique has revealed that certain mutations affecting the N-terminal domain of MRPL45 can impair its integration into the mitoribosomal large subunit .

• Pulse-chase analysis: When combined with metabolic labeling of newly synthesized mitochondrial proteins, MRPL45 antibodies can help assess the functional consequences of assembly defects on mitochondrial translation efficiency.

What is known about MRPL45's membrane interaction mechanism?

MRPL45's interaction with the inner mitochondrial membrane (IMM) represents a critical aspect of mitochondrial translation. Current research findings include:

• Direct membrane interaction: Studies using membrane flotation assays have demonstrated that MRPL45 can interact directly with the IMM, independent of its integration into the mitoribosome . This suggests MRPL45 may anchor the mitoribosome to the membrane during translation of mitochondrially-encoded proteins.

• Structural basis: MRPL45 shows structural similarity to IMM-interacting proteins TIM44 and Mba1, suggesting potential shared mechanisms for membrane association . The protein contains a protruding α-helix with charged amino acids that may participate in membrane interaction, although mutations in this region did not abolish membrane binding .

• N-terminal domain implications: Deletion experiments involving the removal of 117 amino acids from the N-terminal region of MRPL45 (corresponding to the putative membrane-interacting domain of TIM44) affected the protein's integration into the mt-LSU but did not eliminate membrane interaction . This suggests complex determinants for membrane binding beyond simple structural homology.

• Interaction partners: Immunoprecipitation of FLAG-tagged MRPL45 has been performed to identify potential IMM binding partners, although no obvious candidates were detected in mass spectrometry analyses of the immunoprecipitated samples . This indicates that the interaction may be lipid-mediated rather than protein-mediated, or involves transient interactions difficult to capture experimentally.

How do mutations in MRPL45 affect mitochondrial function?

Mutations in MRPL45 and their impact on mitochondrial function have been characterized through several approaches:

• Depletion studies: siRNA-mediated knockdown of MRPL45 confirmed its importance for the stability of the mitoribosomal large subunit and mitochondrial translation . Decreased MRPL45 levels result in impaired mitochondrial protein synthesis, which can lead to energy production deficits.

• Disease associations: Decreased or malfunctioning MRPL45 is linked to mitochondrial disorders, which present with varied clinical symptoms due to compromised energy production . Recently, genomic analyses have identified patients with variants in mitoribosomal genes including MRPL45-related proteins like MRPL39 and MRPL15 .

• Modified MRPL45 expression: Experimental modifications of MRPL45 based on structural similarities with membrane-interacting proteins have shown that certain regions are critical for integration into the mt-LSU. For instance, mutations of charged amino acids on a protruding α-helix of MRPL45 resulted in a protein that could still integrate into the mt-LSU in the absence of endogenous MRPL45, partially rescuing the knockdown phenotype .

• N-terminal truncation: Expression of a mutant MRPL45 lacking 117 amino acids at the N-terminal (corresponding to the putative membrane-interacting domain of TIM44) resulted in poor integration into the mt-LSU, highlighting the importance of this region for proper mitoribosome assembly .

What technical considerations are important when using MRPL45 antibodies for co-immunoprecipitation?

When performing co-immunoprecipitation (co-IP) with MRPL45 antibodies, several technical considerations can improve experimental outcomes:

• Antibody amount optimization: For IP applications, use 0.5-4.0 μg of MRPL45 antibody for 1.0-3.0 mg of total protein lysate . The optimal ratio may need to be determined empirically for specific experimental conditions.

• Cross-linking considerations: When studying transient interactions between MRPL45 and other proteins, particularly membrane proteins, consider using membrane-permeable cross-linking agents prior to cell lysis to stabilize protein complexes.

• Lysis conditions: The choice of lysis buffer can significantly impact co-IP results. For preserving mitoribosomal complexes, gentle non-ionic detergents like digitonin (0.5-1%) or n-dodecyl β-D-maltoside (0.5-1%) are often more suitable than stronger detergents like Triton X-100.

• Salt concentration: The salt concentration in washing buffers affects stringency. Lower salt concentrations (100-150 mM NaCl) preserve weaker interactions, while higher concentrations (300-500 mM NaCl) reduce background but may disrupt physiologically relevant interactions.

• Tagged MRPL45 approach: Studies have successfully used FLAG-tagged MRPL45 for immunoprecipitation, with the added tag not affecting the protein's ability to interact with the membrane or cellular homeostasis . This approach can be valuable when antibody efficiency for native IP is suboptimal.

How can MRPL45 antibodies contribute to understanding mitochondrial disease mechanisms?

MRPL45 antibodies provide valuable tools for investigating mitochondrial disease mechanisms, particularly those involving mitoribosomal dysfunction:

• Diagnostic applications: In patient-derived samples with suspected mitochondrial translation defects, Western blot analysis using MRPL45 antibodies can reveal abnormalities in mitoribosome assembly or stability. Recent multi-omics studies demonstrated that MRPL45 levels, along with other mitoribosomal proteins, show characteristic patterns in patients with mutations in mitoribosomal genes .

• Relative Complex Abundance analysis: This proteomics-based approach can detect defects in OXPHOS disorders with high sensitivity, comparable to or greater than traditional enzymology. MRPL45 antibodies can be used to validate proteomic findings through Western blot or immunofluorescence techniques .

• Tissue-specific investigations: Given the tissue-specific manifestations of many mitochondrial disorders, MRPL45 antibodies can be used for immunohistochemistry to assess mitoribosome integrity across different tissues. The antibodies have been validated for IHC in tissues such as human ovary tumor tissue and mouse brain tissue .

• Exome-unsolved patient studies: For patients with suspected mitochondrial disorders but no clear genetic diagnosis from exome sequencing, immunoblotting with MRPL45 antibodies can provide functional evidence of mitoribosomal defects, potentially guiding further genomic investigations. This approach has been successfully employed to characterize deep intronic variants in mitoribosomal genes that were missed by exome sequencing .

What factors can affect MRPL45 antibody performance in immunofluorescence studies?

Several factors can influence MRPL45 antibody performance in immunofluorescence applications:

• Fixation method: MRPL45 detection is typically performed on PFA-fixed, Triton X-100 permeabilized cells . The fixation duration and concentration should be optimized to maintain mitochondrial morphology while ensuring antibody accessibility.

• Antibody concentration: For immunofluorescence applications, MRPL45 antibodies are typically used at dilutions of 1:200-1:800 . Higher concentrations may increase background signal, while insufficient antibody may result in weak detection.

• Antigen accessibility: As a mitochondrial protein, MRPL45 requires adequate permeabilization of both the cellular and mitochondrial membranes. A sequential permeabilization approach may improve detection in some cell types.

• Signal amplification: For detecting low abundance MRPL45 or in tissues with high autofluorescence, consider using tyramide signal amplification systems or highly sensitive detection methods.

• Co-localization markers: Include established mitochondrial markers (such as TOMM20 or MitoTracker dyes) to confirm the specificity of MRPL45 staining and provide context for its subcellular localization.

How can Western blot protocols be optimized for MRPL45 detection?

For optimal MRPL45 detection by Western blot, consider the following protocol adjustments:

• Sample preparation: MRPL45 has a predicted molecular weight of 35 kDa and is observed at this size in Western blots . For enrichment of the mitochondrial fraction, consider using differential centrifugation or commercially available mitochondrial isolation kits.

• Antibody dilution: MRPL45 antibodies perform well at dilutions ranging from 1:1000 to 1:4000 for Western blot applications . Start with the manufacturer's recommended dilution and adjust as needed based on signal intensity and background.

• Loading controls: Include appropriate mitochondrial loading controls such as VDAC1/Porin or TOMM20 when analyzing isolated mitochondria, or housekeeping proteins like β-actin or GAPDH for whole cell lysates.

• Transfer conditions: As a mitochondrial membrane-associated protein, MRPL45 may require optimized transfer conditions. Consider using PVDF membranes and adding SDS (0.1%) to the transfer buffer to improve transfer efficiency.

• Blocking conditions: Optimize blocking conditions to reduce background while preserving specific signal. BSA-based blocking buffers (3-5%) may perform better than milk-based blockers for some mitochondrial proteins.

• Validated positive controls: Include lysates from cells known to express MRPL45, such as HeLa cells, mouse liver tissue, or SKOV-3 cells, which have been validated for MRPL45 antibody reactivity .

What approaches can resolve inconsistent results with MRPL45 antibodies?

When faced with inconsistent results using MRPL45 antibodies, several troubleshooting approaches may be helpful:

• Antibody validation: Confirm antibody specificity using positive controls (HeLa cells, mouse liver tissue) and negative controls (MRPL45 knockdown samples if available) . Consider testing multiple antibodies targeting different epitopes of MRPL45.

• Sample quality assessment: Mitochondrial proteins are susceptible to degradation. Ensure samples are collected, processed, and stored appropriately with protease inhibitors. Avoid repeated freeze-thaw cycles.

• Protocol standardization: Develop and strictly adhere to standardized protocols for sample preparation, antibody incubation times, washing steps, and detection methods to minimize variability between experiments.

• Environmental factors: Temperature fluctuations during incubation steps can affect antibody binding kinetics. Maintain consistent temperature conditions and consider using temperature-controlled incubators for critical steps.

• Reagent quality control: Regularly check the quality of key reagents including antibodies, detection substrates, and buffers. Consider implementing lot tracking for critical reagents to identify potential batch-related inconsistencies.

• Cross-reactivity assessment: If working with samples from multiple species, validate the antibody's cross-reactivity profile. The MRPL45 antibody has demonstrated reactivity with human, mouse, and rat samples , but efficacy may vary across species.

How can MRPL45 antibodies contribute to studying mitochondrial translation dynamics?

MRPL45 antibodies offer powerful tools for investigating the dynamic processes of mitochondrial translation:

• Proximity labeling approaches: By combining MRPL45 antibodies with proximity labeling techniques (BioID or APEX2), researchers can identify proteins that transiently interact with MRPL45 during mitochondrial translation, providing insights into the dynamic composition of the translation machinery.

• Live-cell imaging: Although direct antibody use is limited to fixed cells, knowledge gained from MRPL45 immunostaining can inform the development of fluorescently tagged MRPL45 constructs for live-cell imaging studies of mitoribosome dynamics.

• Super-resolution microscopy: MRPL45 antibodies can be used with super-resolution techniques like STORM or STED microscopy to visualize the precise spatial organization of mitoribosomes relative to the inner mitochondrial membrane and other mitochondrial compartments.

• Single-molecule translation studies: By combining ribosome profiling techniques with MRPL45 immunoprecipitation, researchers can investigate mitoribosome positioning on specific mitochondrial transcripts to better understand translational regulation.

• Structural studies: MRPL45 antibodies can aid in purifying intact mitoribosomes for structural analyses through cryo-EM, providing insights into how MRPL45 contributes to the ribosome-membrane interface.

What is the relationship between MRPL45 and mitochondrial disease pathology?

MRPL45's role in mitochondrial disease pathology is becoming increasingly apparent: