mrpl-18 Antibody

What is MRPL-18 and what are its primary functions?

MRPL-18 (also known as Large ribosomal subunit protein uL18m, L18mt, or MRP-L18) is a mitochondrial ribosomal protein that facilitates efficient translation of genetic material into functional proteins. It collaborates with other ribosomal proteins and rRNA to maintain mitochondrial function . Beyond its mitochondrial role, MRPL-18 has an unexpected function in the mammalian cytosolic stress response. Research has revealed that MRPL-18 generates a cytosolic isoform that incorporates into the 80S ribosome and facilitates ribosome engagement on mRNAs selected for translation during stress . This dual functionality makes MRPL-18 a fascinating target for researchers studying mitochondrial biology, translation mechanisms, and cellular stress responses.

What types of MRPL-18 antibodies are available for research?

Several MRPL-18 antibodies are available for research applications, primarily differing in host species, clonality, and validated applications:

These antibodies have been validated through various techniques including Western blotting, immunohistochemistry, and ELISA. When selecting an antibody, consider your specific experimental needs, target species, and preferred application methodology .

How do the mitochondrial and cytosolic isoforms of MRPL-18 differ?

The MRPL-18 transcript contains three translation initiation sites (TIS): an upstream TIS (uTIS), the annotated start codon (aTIS), and a downstream TIS (dTIS) . The cytosolic isoform is produced through alternative translation initiation at a CUG codon (dTIS) that is located immediately after the mitochondrial targeting signal (MTS) . This results in a protein lacking the MTS, which remains in the cytosol rather than being imported into mitochondria.

While the mitochondrial isoform functions in mitochondrial translation as part of the mitochondrial ribosome, the cytosolic isoform plays a distinct role in the cellular stress response. During stress conditions, the cytosolic MRPL-18 incorporates into the 80S ribosome and facilitates the translation of stress-responsive mRNAs, particularly those encoding heat shock proteins (HSPs) . This dual functionality represents an elegant mechanism by which cells adapt to stress conditions by repurposing a mitochondrial protein for cytosolic functions.

What are the optimal conditions for Western blot analysis using MRPL-18 antibodies?

For optimal Western blot analysis using MRPL-18 antibodies, researchers should consider the following methodological approach:

• Sample preparation: Prepare cell or tissue lysates using standard protocols. For most applications, 15-25 μg of total protein is recommended based on published methods .

• Gel electrophoresis: Use 10% polyacrylamide gels for optimal separation of MRPL-18, which has a predicted molecular weight of 21 kDa but is typically observed at 18 kDa on SDS-PAGE .

• Transfer conditions: Transfer proteins to nitrocellulose membranes at 100 V for 2 hours as described in published protocols .

• Antibody dilutions:

  • Mouse polyclonal antibody (ab67844): Use at 1:500 dilution

  • Rabbit polyclonal antibody (A305677): Use at 1:500-1:2,000 dilution

  • Rabbit polyclonal antibody (orb355655): Use at 1:500-2,000 dilution

• Incubation conditions: Incubate with primary antibody overnight at 4°C, followed by appropriate secondary antibody incubation for 1 hour at room temperature .

• Expected results: The predicted band size for MRPL-18 is 21 kDa, though actual observed band size is often around 18 kDa , likely corresponding to the processed form after MTS cleavage.

When analyzing MRPL-18 by Western blot, it's important to note that you may observe multiple bands representing different isoforms - the full-length precursor and the processed form lacking the MTS .

How can I optimize immunohistochemistry protocols for MRPL-18 detection?

For researchers conducting immunohistochemistry (IHC) with MRPL-18 antibodies:

• Tissue preparation: Use standard formalin fixation and paraffin embedding procedures. Published studies have successfully used MRPL-18 antibodies on human kidney and testis tissue sections .

• Antigen retrieval: While specific conditions aren't detailed in the search results, standard heat-induced epitope retrieval in citrate buffer (pH 6.0) is likely appropriate based on general protocols for mitochondrial proteins.

• Antibody dilution: For the orb355655 antibody, use a dilution range of 1:20-1:200 for IHC-P applications .

• Detection system: Use an appropriate detection system compatible with the host species of your primary antibody (e.g., HRP-conjugated anti-rabbit secondary antibody for rabbit primary antibodies).

• Controls: Include positive controls (tissues known to express MRPL-18, such as kidney or testis) and negative controls (omitting primary antibody) to validate staining specificity.

• Expected staining pattern: MRPL-18 should primarily show a mitochondrial distribution pattern, appearing as cytoplasmic granular staining in cells with high mitochondrial content.

What approaches are recommended for validating MRPL-18 antibody specificity?

To ensure experimental rigor, researchers should validate MRPL-18 antibody specificity through multiple complementary approaches:

• Western blot analysis: Confirm that the antibody detects a protein of the expected molecular weight (18-21 kDa) in positive control samples. Multiple cell lines have been validated including U251, THP-1, PC-3, and U87 .

• Knockdown/knockout validation: Perform siRNA knockdown or CRISPR/Cas9 knockout of MRPL-18 and demonstrate reduced or absent signal compared to control cells.

• Recombinant protein controls: Use recombinant MRPL-18 protein as a positive control, particularly useful when working with new tissue types or experimental conditions.

• Subcellular localization: Confirm appropriate mitochondrial localization through subcellular fractionation or co-localization studies with established mitochondrial markers.

• Cross-reactivity assessment: Test the antibody against related proteins or in species not claimed to have reactivity to evaluate potential cross-reactivity.

• Multiple antibody validation: When possible, confirm key findings using multiple antibodies targeting different epitopes of MRPL-18.

This systematic validation approach ensures that experimental observations are genuinely attributable to MRPL-18 rather than non-specific interactions or cross-reactivity.

How can MRPL-18 antibodies be used to study stress-induced translational control?

MRPL-18 antibodies provide powerful tools for investigating stress-induced translational control mechanisms:

• Detecting stress-induced isoform shifts: Western blot analysis can be used to monitor the increased production of the cytosolic MRPL-18 isoform following stress treatments. During heat shock, researchers have observed a modest increase in full-length MRPL-18 and a more pronounced increase in the CUG-initiated cytosolic isoform .

• Polysome profiling: MRPL-18 antibodies can be used in conjunction with polysome profiling to detect the incorporation of cytosolic MRPL-18 into actively translating ribosomes during stress. Endogenous MRPL-18 has been recovered from polysome fractions after heat shock stress, with this redistribution being sensitive to the translation inhibitor cycloheximide .

• Co-immunoprecipitation studies: Using MRPL-18 antibodies for co-immunoprecipitation experiments can identify stress-specific protein-protein interactions. Endogenous MRPL-18 has been precipitated from stressed cells using antibodies against cytosolic ribosomal proteins RPL4 and RPS6, confirming its association with the 80S ribosome during stress .

• Reporter assays: Combine MRPL-18 antibodies with reporter constructs (such as luciferase reporters) to study the functional consequences of MRPL-18 manipulation on stress-responsive gene expression .

• Immunofluorescence microscopy: Track the subcellular redistribution of MRPL-18 during stress responses through immunofluorescence staining combined with mitochondrial and ribosomal markers.

These approaches have revealed that MRPL-18 plays a critical role in forming "hybrid" ribosomes that are responsible for translational regulation during the cytosolic stress response, particularly in the synthesis of heat shock proteins .

What methods can be used to study the role of MRPL-18 in mitochondrial translation and ribosome assembly?

To investigate MRPL-18's role in mitochondrial translation and ribosome assembly:

• Mitochondrial ribosome isolation and analysis: Use MRPL-18 antibodies to track its incorporation into mitochondrial ribosomes through density gradient centrifugation and Western blot analysis. Exogenous MRPL-18 has been shown to incorporate into mitochondrial ribosomes, co-sedimenting with other mitochondrial ribosomal proteins .

• Import assays: Study the import of MRPL-18 into mitochondria using in vitro import assays with radiolabeled precursors or fluorescently tagged constructs, followed by detection with MRPL-18 antibodies.

• Interaction studies with 5S rRNA: Investigate MRPL-18's function as a mitochondrial import factor for cytosolic 5S rRNA through RNA immunoprecipitation or electrophoretic mobility shift assays (EMSAs) using MRPL-18 antibodies. MRPL-18, together with thiosulfate sulfurtransferase (TST), acts as a mitochondrial import factor for cytosolic 5S rRNA .

• Mitochondrial translation assays: Assess the impact of MRPL-18 depletion or mutation on mitochondrial protein synthesis using metabolic labeling approaches (e.g., [35S]-methionine incorporation).

• Blue native PAGE (BN-PAGE): Analyze mitochondrial ribosome assembly states following MRPL-18 manipulation through BN-PAGE followed by Western blotting with MRPL-18 antibodies.

Interestingly, while MRPL-18 is essential for mitochondrial ribosome function, knockdown experiments have shown minimal effects on mitochondrial function despite significantly dampening cytosolic heat shock protein expression , suggesting potential compensatory mechanisms within mitochondria.

How can MRPL-18 be studied in the context of diabetes and cancer research?

Recent research has implicated MRPL-18 in both diabetes and cancer pathophysiology, offering new research directions:

• Expression analysis in disease states: Quantify MRPL-18 levels in diabetic and cancer tissues using ELISA or Western blot approaches. Studies have shown elevated levels of MRPL-18 in normal colon epithelial cells exposed to high-glucose conditions, suggesting a link to diabetes pathophysiology .

• Glucose regulation studies: Investigate how glucose levels affect MRPL-18 expression and subcellular distribution using a combination of ELISA, Western blot, and immunofluorescence techniques under controlled glucose conditions .

• Cancer cell line panels: Analyze MRPL-18 expression across diverse cancer cell lines (such as U251, THP-1, PC-3, and U87) to identify cancer-specific expression patterns .

• Functional genomics approaches: Employ CRISPR/Cas9 or RNAi techniques to modulate MRPL-18 expression in diabetes or cancer models, followed by phenotypic characterization.

• Translational stress response in cancer: Investigate whether the stress-responsive functions of cytosolic MRPL-18 contribute to cancer cell survival under adverse conditions through selective translation of pro-survival factors.

• Clinical correlation studies: Correlate MRPL-18 expression levels with clinical outcomes in diabetes or cancer patients through immunohistochemical analysis of tissue microarrays.

These approaches may reveal whether MRPL-18 represents a potential therapeutic target or biomarker in diabetes-associated carcinogenesis pathways .

What are common challenges when detecting MRPL-18 by Western blot and how can they be addressed?

Researchers may encounter several challenges when detecting MRPL-18 by Western blot:

• Multiple band detection: MRPL-18 can appear as multiple bands representing different isoforms (precursor vs. processed forms). The predicted band size is 21 kDa, but the observed band is often around 18 kDa . To disambiguate these bands:

  • Use positive controls with known MRPL-18 expression

  • Compare with recombinant MRPL-18 protein standards

  • Perform subcellular fractionation to separate mitochondrial and cytosolic fractions

• Low signal intensity: MRPL-18 may be expressed at relatively low levels under basal conditions, particularly the cytosolic isoform. To improve detection:

  • Increase protein loading (30-50 μg)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use enhanced chemiluminescence (ECL) detection systems

  • Consider stress treatments (e.g., heat shock) which increase MRPL-18 expression

• Non-specific bands: To reduce background and non-specific binding:

  • Optimize blocking conditions (5% non-fat milk or BSA)

  • Increase wash stringency and duration

  • Titrate primary antibody concentration

  • Pre-adsorb antibody with non-specific proteins

• Inconsistent results across cell types: Different cell types may express varying levels of MRPL-18 isoforms. Western blot analysis has been successfully performed on various cell lines including U251, THP-1, PC-3, and U87 , as well as human liver tissue .

How can researchers distinguish between mitochondrial and cytosolic MRPL-18 isoforms?

Distinguishing between mitochondrial and cytosolic MRPL-18 isoforms requires specific experimental approaches:

• Subcellular fractionation: Separate mitochondrial and cytosolic fractions through differential centrifugation or commercial fractionation kits, followed by Western blot analysis with MRPL-18 antibodies. The mitochondrial isoform should be enriched in mitochondrial fractions, while the cytosolic isoform will be present in the cytosolic fraction.

• Stress induction: Apply heat shock or other stress treatments to induce the cytosolic isoform. The cytosolic MRPL-18 shows increased expression following stress, while mitochondrial MRPL-18 levels remain relatively stable .

• Co-immunoprecipitation with compartment-specific markers: Use antibodies against mitochondrial ribosomal proteins (for mitochondrial MRPL-18) or cytosolic ribosomal proteins like RPL4 or RPS6 (for cytosolic MRPL-18) to co-immunoprecipitate the respective isoforms .

• Size discrimination: The full-length precursor MRPL-18 (containing the MTS) is slightly larger than the processed mitochondrial form or the cytosolic isoform. High-resolution SDS-PAGE may separate these forms based on subtle size differences .

• Isoform-specific mutants: Generate and express MRPL-18 constructs with mutations in specific initiation codons. For example, MRPL18(ATG) lacks both the 5'UTR and MTS region, while MRPL18(C14A) uses only the CUG initiator to produce the cytosolic isoform .

These approaches help researchers accurately attribute experimental observations to the appropriate MRPL-18 isoform and corresponding cellular function.

What considerations are important when interpreting MRPL-18 expression data in different experimental contexts?

When interpreting MRPL-18 expression data across experimental contexts, researchers should consider:

• Stress conditions: MRPL-18 expression, particularly the cytosolic isoform, is responsive to cellular stress. Heat shock significantly increases cytosolic MRPL-18 levels, with the CUG-initiated isoform showing the strongest responsiveness despite low basal levels in unstressed cells . Therefore, experimental stress conditions must be carefully controlled and reported.

• Cell type specificity: MRPL-18 expression may vary considerably between cell types. Studies have examined MRPL-18 in various cell lines including U251, THP-1, PC-3, and U87 , as well as in human kidney and testis tissues . Comparisons across cell types should acknowledge these potential differences.

• Metabolic state: High glucose conditions can alter MRPL-18 expression, as demonstrated in colon epithelial cells . The metabolic state of experimental systems should be considered when interpreting expression data.

• Translational vs. transcriptional regulation: MRPL-18 expression is regulated at both transcriptional and translational levels. The cytosolic isoform is produced through alternative translation initiation , highlighting the importance of protein-level analysis alongside transcriptional studies.

• Technical variables: Different antibodies may preferentially detect certain epitopes or isoforms of MRPL-18. When comparing data across studies, consider whether the same antibody was used and at what dilution.

• Functional readouts: Changes in MRPL-18 expression should be correlated with functional outcomes, such as mitochondrial translation efficiency, stress response activation, or alterations in heat shock protein expression .

By considering these factors, researchers can more accurately interpret MRPL-18 expression data and its biological significance in different experimental contexts.