MRPL52 Antibody

What is MRPL52 and what is its cellular function?

MRPL52 is a nuclear-encoded mitochondrial ribosomal protein that participates in protein synthesis within the mitochondrion. It is a component of the 39S large subunit of mitochondrial ribosomes (mitoribosomes) . Unlike prokaryotic ribosomes, mammalian mitoribosomes have an estimated 75% protein to rRNA composition, and MRPL52 specifically has no bacterial homolog . The protein is essential for translating mitochondrial-encoded genes that are critical for oxidative phosphorylation and cellular respiration. Through these pathways, MRPL52 supports cellular energy metabolism and helps in the production of essential proteins for mitochondrial respiration .

While the calculated molecular weight of MRPL52 is approximately 14 kDa based on its 123 amino acid sequence , Western blot experiments typically detect the protein at 18-21 kDa . This discrepancy between predicted and observed molecular weight may be attributed to post-translational modifications, relative charges, or other experimental factors affecting protein migration in SDS-PAGE . Researchers should be aware of this difference when interpreting their Western blot results.

What species reactivity do MRPL52 antibodies typically exhibit?

Most commercially available MRPL52 antibodies demonstrate reactivity with:

• Human

• Mouse

• Rat

This cross-species reactivity is documented across multiple antibody sources , making these antibodies versatile tools for comparative studies across mammalian models. Some antibodies have been specifically cited in publications using human and mouse samples .

Where is MRPL52 localized within cells?

As a mitochondrial ribosomal protein, MRPL52 is primarily localized to mitochondria. Immunofluorescence studies show positivity in mitochondria but not in the nucleus or nucleoli . Positive IF/ICC detection has been reported in various cell lines, including HepG2 cells and U-251MG cells . When performing co-localization studies, researchers should consider using mitochondrial markers such as COX IV as controls to confirm the mitochondrial localization of MRPL52 .

How is MRPL52 expression regulated under hypoxic conditions?

Research indicates that MRPL52 expression is upregulated under hypoxic conditions in a HIF-1 (Hypoxia-Inducible Factor-1) dependent manner . Li et al. (2021) identified potent hypoxia response elements in the promoter region of MRPL52 and validated HIF-1 binding through chromatin immunoprecipitation and luciferase reporter assays . This hypoxia-induced upregulation appears to be an adaptive mechanism that allows cancer cells, particularly breast cancer cells, to adjust their mitochondrial function to survive in the hypoxic tumor microenvironment . This regulation mechanism represents an important consideration when studying MRPL52 in cancer models, especially when designing experiments involving oxygen tension manipulation.

What role does MRPL52 play in cancer progression?

MRPL52 has been implicated in several cancer-related processes, particularly in breast cancer under hypoxic conditions:

• Apoptosis resistance: MRPL52 suppresses apoptosis in hypoxic breast cancer cells by facilitating mitophagy, which delays the vicious cycle between mitochondrial damage and excessive ROS generation .

• Metastasis promotion: MRPL52 enhances the migration and invasion capabilities of cancer cells through activation of the ROS-Notch1-Snail signaling pathway .

• ROS regulation: MRPL52 maintains reactive oxygen species (ROS) levels within a range that promotes tumorigenic signaling without triggering cytotoxicity .

• EMT facilitation: By mediating the ROS-Notch1-Snail pathway, MRPL52 promotes epithelial-mesenchymal transition, a critical step in cancer metastasis .

Clinical evidence shows significant correlation between MRPL52 upregulation and higher metastatic risk and poorer clinicopathological characteristics in breast cancer specimens . These findings suggest MRPL52 could be a potential therapeutic target for metastatic cancer treatment.

As a component of the mitochondrial ribosome large subunit (39S), MRPL52 participates in the translation of proteins encoded by mitochondrial DNA . Mitochondrial ribosomes differ significantly from cytosolic ribosomes in their composition, with an estimated 75% protein to rRNA ratio (compared to prokaryotic ribosomes where this ratio is reversed) . MRPL52's precise role in the translation mechanism may be unique since it has no bacterial homolog .

The protein is essential for proper mitochondrial function, as it contributes to the synthesis of components of the electron transport chain necessary for oxidative phosphorylation . Recent research with CRISPR screens has implicated MRPL52 (Mrpl52 in mice) in enabling early tissue-resident memory T cell (TRM) formation, suggesting additional roles beyond basic mitochondrial protein synthesis .

What approaches can be used to validate MRPL52 antibody specificity?

To ensure experimental rigor, researchers should validate MRPL52 antibody specificity using multiple approaches:

• Positive controls: Use tissues known to express MRPL52, such as kidney, spleen, or testis for IHC applications .

• Knockdown validation: Compare antibody signal between wild-type cells and those with MRPL52 knockdown (using siRNAs or CRISPR-Cas9) .

• Molecular weight verification: Confirm that the observed band in Western blot appears at the expected 18-21 kDa range .

• Subcellular localization: Verify mitochondrial localization using co-staining with established mitochondrial markers like COX IV or MitoTracker .

• Competition assays: For immunoprecipitation experiments, include competition controls with MRPL52 recombinant protein or peptide to demonstrate binding specificity .

• Multiple antibody validation: When possible, confirm results using antibodies targeting different epitopes of MRPL52 .

How can researchers optimize co-localization studies involving MRPL52?

For effective co-localization of MRPL52 with other mitochondrial proteins:

• Antibody selection: Use antibodies raised in different host species to avoid cross-reactivity when performing double immunostaining .

• Fixation optimization: 4% PFA fixation for 20 minutes has been successfully used in multiple studies .

• Permeabilization: 0.2% Triton X-100 in PBS for 5 minutes effectively permeabilizes cells while preserving mitochondrial structure .

• Mitochondrial labeling: Consider pre-labeling with MitoTracker Green FM (1:1000 dilution) before fixation for co-localization studies .

• Confocal imaging: Use Z-stack imaging with appropriate laser lines (e.g., 402, 488, and 561 nm) to capture the three-dimensional distribution of MRPL52 in mitochondria .

• Controls: Include single-stained controls to account for potential bleed-through between channels.

What is known about MRPL52's role in T cell development?

Recent research using CRISPR screens has revealed that MRPL52 (Mrpl52 in mice) enables early tissue-resident memory T cell (TRM) formation . This finding suggests a previously unappreciated role for MRPL52 in immune cell development and function. The study indicated that genetic interaction screening identified Mrpl52 as a critical factor that enables early TRM cell formation . This finding expands our understanding of MRPL52 beyond its basic mitochondrial functions and into immunological processes, opening new avenues for research in immunometabolism.

How can weak MRPL52 signals be improved in Western blot experiments?

If you encounter weak MRPL52 signal in Western blot:

• Increase protein loading: Try 30-50 μg of total protein from mitochondrial-enriched fractions.

• Optimize antibody concentration: Adjust within the recommended range (1:250-1:2000) based on signal strength .

• Extend antibody incubation: Consider overnight incubation at 4°C for primary antibody.

• Use appropriate controls: Include mitochondrial markers (like COX IV) as loading controls rather than cytosolic proteins .

• Sample preparation: Use buffers containing protease inhibitors and avoid excessive freeze-thaw cycles to preserve protein integrity.

• Enhanced detection: Use high-sensitivity chemiluminescence detection reagents for visualizing low-abundance proteins.

What considerations are important when designing experiments to study MRPL52 under hypoxic conditions?

When studying MRPL52 under hypoxic conditions:

• Oxygen control: Use a validated hypoxic chamber capable of maintaining stable 1% O2 and 5% CO2 conditions .

• Time course: Consider both acute (24h) and chronic (48-72h) hypoxia exposures, as MRPL52 responses may differ .

• HIF-1α validation: Include HIF-1α detection as a positive control for hypoxic response .

• ROS measurement: Incorporate ROS detection methods to correlate with MRPL52 expression changes .

• Genetic manipulation: Use HIF-1α knockdown to confirm the dependency of MRPL52 upregulation on this transcription factor .

• Normalization: Use hypoxia-stable reference genes when performing qPCR analysis of MRPL52 expression.

• Functional endpoints: Assess mitochondrial function (using Seahorse analyzers) alongside MRPL52 expression changes .