MRPS18B Antibody

What is MRPS18B and what is its biological significance?

MRPS18B is a component of the small subunit of the mitochondrial ribosome that plays a crucial role in protein synthesis within mitochondria. It's one of three variants of the S18 protein family (alongside MRPS18a and MRPS18c) that are products of separate genes with only 25-30% sequence identity between them . The protein is essential for normal mitochondrial function, and its dysregulation has been linked to various diseases, including cancer and neurodegenerative disorders . Recent research has revealed that MRPS18B may have functions beyond mitochondrial translation, potentially participating in cellular transformation processes .

What are the validated applications for MRPS18B antibodies in research?

MRPS18B antibodies have been validated for multiple research applications:

These applications allow researchers to study MRPS18B expression levels, localization patterns, and interactions with other proteins in various experimental contexts .

What controls should be included when using MRPS18B antibodies in experimental procedures?

When working with MRPS18B antibodies, several controls are essential to ensure reliable results:

• Positive controls: Cell lines or tissues known to express MRPS18B, such as breast cancer cell lines like MCF7, which have been demonstrated to express detectable levels of MRPS18B .

• Negative controls:

  • Primary antibody omission control to assess secondary antibody specificity

  • Tissues or cells with MRPS18B knockdown/knockout

  • Isotype controls matching the primary antibody's host species and isotype (typically rabbit IgG)

• Loading controls: For Western blot, include mitochondrial markers (e.g., VDAC) when examining mitochondrial fractions, or standard housekeeping proteins for whole-cell lysates .

• Specificity validation: Far western blot analysis can be used to confirm antibody specificity to MRPS18B, particularly when evaluating conformational epitope recognition, as demonstrated in studies with related proteins like MRPS18a .

How can researchers optimize MRPS18B detection in Western blot applications?

For optimal MRPS18B detection in Western blot:

• Sample preparation: Prepare mitochondrial fractions to enrich for MRPS18B, which enhances detection sensitivity. Use gentle lysis buffers containing protease inhibitors to preserve protein integrity .

• Protein denaturation: Some MRPS18B antibodies recognize conformational epitopes. If standard Western blot protocols yield poor results, try modified denaturation conditions or far Western blot techniques, which allow renaturation of proteins on the membrane .

• Blocking optimization: Use 5% non-fat dry milk or BSA in TBS-T for blocking. Test both as MRPS18B antibody performance can vary depending on blocking agent.

• Antibody dilution optimization: Begin with the manufacturer's recommended dilution (typically 1:500 for Western blot), then perform a dilution series to determine optimal concentration for your specific sample type .

• Signal enhancement: For low abundance samples, consider using enhanced chemiluminescence detection systems with longer exposure times or signal amplification methods.

• Molecular weight verification: MRPS18B typically appears at approximately 28kDa, but validation with recombinant protein standards is recommended as post-translational modifications may affect migration .

What are the best practices for using MRPS18B antibodies in immunohistochemistry?

For optimal immunohistochemical detection of MRPS18B:

• Fixation: Use 10% neutral buffered formalin for tissue fixation. Overfixation can mask epitopes, so limit fixation time to 24-48 hours .

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is generally effective. For MRPS18B, heat retrieval at 95-98°C for 15-20 minutes typically yields optimal results.

• Antibody dilution: Start with manufacturer's recommended dilution (1:200-1:500) and optimize based on signal-to-noise ratio .

• Detection systems: Polymer-based detection systems generally provide better sensitivity than standard ABC methods for mitochondrial proteins like MRPS18B.

• Counterstaining: Use light hematoxylin counterstaining to avoid obscuring mitochondrial staining patterns.

• Interpretation: MRPS18B typically shows granular cytoplasmic staining corresponding to mitochondrial localization. Nuclear staining may be observed in some cancer cells, potentially indicating non-canonical functions .

How has MRPS18B been implicated in cancer research, and what methods are used to study this relationship?

MRPS18B has been implicated in oncogenesis through several research approaches:

• Expression studies: Research has demonstrated differential expression of MRPS18B in cancer tissues compared to normal tissues. Similar to the related protein MRPS18a, MRPS18B may show increased expression in cancer cells, particularly in breast cancer .

• Functional studies: Overexpression of MRPS18B in human breast and renal cancer cell lines has been shown to result in the appearance of multinucleated cells, suggesting a potential role in abnormal cell division and cancer progression .

• Transformation potential: Studies have shown that overexpression of human MRPS18B in primary rat embryonic fibroblasts leads to immortalization. The resulting cell lines adopt several cancer-like characteristics, including loss of contact inhibition and anchorage-independent growth .

• Stem cell marker association: MRPS18B overexpression has been associated with the expression of various stem cell markers, potentially linking it to cancer stem cell properties .

• Metabolic alterations: As a mitochondrial ribosomal protein, MRPS18B may play a role in the metabolic reprogramming observed in cancer cells (the Warburg effect), similar to other mitochondrial ribosomal proteins like MRPL28 .

To study these relationships, researchers employ techniques including:

• Immunohistochemistry to compare expression levels between normal and cancer tissues

• Western blot analysis of different cellular fractions

• Overexpression and knockdown studies to assess functional consequences

• Colocalization studies with other cancer markers

What are the considerations for using MRPS18B antibodies in comparative studies between normal and disease states?

When using MRPS18B antibodies to compare normal and disease tissues:

How can researchers address non-specific binding when using MRPS18B antibodies?

Non-specific binding is a common challenge when working with MRPS18B antibodies. To address this issue:

• Antibody selection: Choose antibodies that have been affinity-purified against the immunogen, as these generally show higher specificity .

• Blocking optimization: Increase blocking time or concentration (5-10% normal serum from the same species as the secondary antibody). For Western blots, compare BSA vs. non-fat dry milk as blocking agents to determine which reduces background more effectively.

• Washing protocols: Implement more stringent washing steps with higher salt concentration in TBS-T (up to 500mM NaCl) to reduce non-specific ionic interactions.

• Antibody pre-adsorption: For tissue with high background, pre-adsorb the antibody with tissue powder from the species being studied to remove antibodies that bind to common epitopes.

• Epitope-specific antibodies: When available, select antibodies targeting unique regions of MRPS18B. The N-terminal region (amino acids 1-94) appears to be a good target for specific recognition based on available antibodies .

• Cross-reactivity testing: If working with non-human samples, test antibodies on multiple species' samples to confirm specificity, as MRPS18B antibodies may show different cross-reactivity patterns despite predicted sequence homology .

How should researchers interpret conflicting results between different MRPS18B antibody clones?

When faced with conflicting results between different MRPS18B antibody clones:

• Epitope mapping: Determine the specific epitopes recognized by each antibody. Different antibodies may recognize different regions of MRPS18B, which could be differentially accessible depending on protein conformation, interactions, or post-translational modifications .

• Validation with recombinant protein: Test each antibody against recombinant MRPS18B protein to confirm basic recognition capabilities and compare relative affinities.

• Knockdown/knockout controls: Validate each antibody using MRPS18B knockdown or knockout samples to confirm specificity and rule out off-target binding.

• Application-specific performance: Some antibodies may perform well in certain applications but poorly in others. For example, some MRPS18B antibodies recognize conformational epitopes and work in far Western blot but not standard Western blot protocols .

• Isoform specificity: Consider whether conflicting results might reflect detection of different MRPS18B isoforms or related family members (MRPS18a, MRPS18c) .

• Complementary approaches: Use alternative methods to validate findings, such as mass spectrometry or RNA-level analyses (qPCR, RNA-seq) to corroborate protein-level observations.

How can MRPS18B antibodies contribute to studies on mitochondrial dysfunction in neurodegenerative diseases?

MRPS18B antibodies can provide valuable insights into mitochondrial dysfunction in neurodegenerative diseases through several approaches:

• Expression level analysis: Quantitative analysis of MRPS18B expression in affected brain regions compared to controls can indicate alterations in mitochondrial ribosome composition, potentially revealing disease-specific changes in mitochondrial translation .

• Colocalization studies: Dual immunofluorescence with MRPS18B antibodies and markers of mitochondrial stress or damage can reveal spatial relationships between mitochondrial ribosome alterations and pathological features.

• Mitochondrial morphology correlation: Using MRPS18B as a mitochondrial marker in combination with imaging of mitochondrial networks can help correlate changes in mitochondrial translation machinery with alterations in mitochondrial dynamics common in neurodegenerative diseases.

• Patient-derived cell models: MRPS18B antibodies can be used to assess mitochondrial translation capacity in patient-derived neurons or glial cells, potentially identifying cell type-specific vulnerabilities.

• Therapeutic response monitoring: In experimental therapeutic interventions targeting mitochondrial function, MRPS18B antibodies can help monitor changes in mitochondrial translation machinery as a response biomarker.

• Post-mortem tissue analysis: Immunohistochemical studies using MRPS18B antibodies on post-mortem brain tissue can reveal disease-specific alterations in mitochondrial ribosome distribution and abundance across different neural cell types .

What potential exists for MRPS18B as a biomarker in clinical research?

MRPS18B shows promise as a biomarker in several clinical research contexts:

• Cancer diagnostics: Given the observed upregulation of MRPS18B family proteins in cancer cells, particularly breast cancer, MRPS18B could serve as part of a biomarker panel for cancer detection or subtyping .

• Prognostic indicator: Expression levels of MRPS18B might correlate with disease progression or treatment response, similar to other mitochondrial ribosomal proteins that have been linked to cancer outcomes .

• Therapeutic target identification: MRPS18B antibodies can help identify patient populations that might benefit from therapies targeting mitochondrial function, by quantifying MRPS18B expression levels or localization patterns.

• Tissue microarray studies: High-throughput analysis of MRPS18B expression across large patient cohorts using tissue microarrays and validated antibodies could reveal correlations with clinical parameters and outcomes.

• Liquid biopsy development: MRPS18B antibodies could potentially be used in the development of liquid biopsy approaches, detecting MRPS18B in circulating tumor cells or extracellular vesicles as a cancer biomarker.

• Mitochondrial disease classification: For inherited or acquired mitochondrial disorders, MRPS18B expression patterns might help classify disease subtypes or severity, particularly in conditions affecting mitochondrial translation .