MRPL9 Antibody

What is MRPL9 and why is it significant in research contexts?

MRPL9 (mitochondrial ribosomal protein L9) is a key component of the mitochondrial ribosomal complex essential for the translation of mitochondrial-encoded proteins. It integrates into the mitochondrial ribosome machinery and plays a crucial role in mitochondrial function . Recent research has identified MRPL9 as being highly expressed in certain cancers, including papillary thyroid cancer (PTC) and liver cancer, where it promotes cell proliferation and migration . The protein's significance extends to its potential as a biomarker for cancer monitoring and treatment, particularly in PTC .

What applications are MRPL9 antibodies validated for?

MRPL9 antibodies have been validated for multiple research applications:

These applications have been validated across multiple commercial antibodies with demonstrated reactivity against human, mouse, and rat MRPL9 .

What is the molecular weight of MRPL9 and what band sizes should I expect in Western blotting?

When analyzing MRPL9 via Western blotting, researchers should note the following:

• Calculated molecular weight: 30 kDa

• Observed molecular weight: 26-28 kDa

This discrepancy between calculated and observed weights is consistent across multiple antibody validation studies and may reflect post-translational modifications or structural characteristics of the protein . When troubleshooting Western blots, this 26-28 kDa range should be considered the primary target band.

How can I verify antibody specificity for MRPL9 when conducting cross-reactivity studies?

Antibody cross-reactivity is a significant concern in MRPL9 research, as highlighted by studies on antibody specificity . To verify specificity:

• Conduct validation with positive and negative controls: Include cell lines known to express MRPL9 (e.g., HeLa, HepG2, MCF-7, PC-3) as positive controls .

• Use multiple antibodies targeting different epitopes: Compare results from antibodies recognizing different regions of MRPL9, such as N-terminal vs. C-terminal domains .

• Implement knockdown/knockout validation: Utilize MRPL9 knockdown systems to confirm signal reduction in Western blot and immunostaining applications .

• Perform peptide competition assays: Pre-incubate the antibody with the immunizing peptide to verify that specific binding is blocked.

• Compare observed vs. expected molecular weight patterns: Examine whether the observed band pattern matches expected sizes (26-28 kDa), as discrepancies may indicate cross-reactivity .

The importance of these validation steps is underscored by research showing that antibody cross-reactivity can account for widespread mapping artifacts in transcriptome studies .

What are the optimal conditions for MRPL9 detection in immunohistochemistry?

For optimal MRPL9 immunohistochemical detection:

• Antigen retrieval optimization: Most protocols recommend TE buffer at pH 9.0, though citrate buffer at pH 6.0 can be used as an alternative . This step is critical as improper antigen retrieval is a common cause of false negatives.

• Antibody dilution range: Begin with a 1:50-1:500 dilution range, with many protocols finding 1:200 to be optimal for paraffin-embedded tissues .

• Positive control tissues: Use breast cancer tissue samples, which have been validated as positive controls for MRPL9 antibodies in multiple studies .

• Detection system selection: Enzyme-based detection systems (HRP/DAB) provide excellent signal-to-noise ratio for MRPL9 detection in tissue samples.

• Cellular localization expectations: Anticipate primarily mitochondrial localization patterns, with potential nuclear signals in certain cancer tissues .

Published validation data shows successful MRPL9 detection in human breast cancer tissue with these methodological approaches .

How do I design experiments to study MRPL9 interaction with GGCT and its role in the MAPK/ERK pathway?

Recent research has identified a critical interaction between MRPL9 and GGCT (γ-glutamylcyclotransferase) that modulates the MAPK/ERK pathway in papillary thyroid cancer . To investigate this interaction:

• Co-immunoprecipitation (Co-IP) approach:

  • Use anti-GGCT antibodies for immunoprecipitation followed by Western blotting with anti-MRPL9 antibodies

  • The reverse approach (IP with anti-MRPL9, WB with anti-GGCT) should also be performed for confirmation

  • Include appropriate IgG controls to verify specificity

• Immunofluorescence co-localization studies:

  • Implement dual immunofluorescence staining for MRPL9 and GGCT

  • Use confocal microscopy to analyze subcellular co-localization patterns

  • Compare normal and cancer tissue samples for differential co-localization patterns

• Functional validation through knockdown experiments:

  • Design experiments with individual knockdowns of MRPL9 and GGCT, as well as double knockdowns

  • Analyze MAPK/ERK pathway activation through phosphorylation status of key components

  • Measure cellular outcomes including proliferation and migration capacity

Research has shown that dual knockdown of MRPL9 and GGCT resulted in stronger inhibition of tumor cell proliferation and migration than single knockdown of either protein alone, suggesting synergistic effects .

Why am I detecting multiple bands in Western blots with MRPL9 antibodies?

Multiple bands in MRPL9 Western blots may arise from several sources:

• Isoform detection: While the canonical MRPL9 protein is expected at 26-28 kDa, additional bands may represent alternative splicing variants or processed forms of the protein.

• Post-translational modifications: MRPL9 may undergo modifications affecting mobility. Researchers should consider using phosphatase treatment or other modification-specific approaches to investigate this possibility.

• Cross-reactivity with similar proteins: Some antibodies may cross-react with other mitochondrial ribosomal proteins with similar epitopes. Validation with knockout/knockdown controls is essential to determine specificity .

• Protein degradation: Partial degradation during sample preparation can generate fragments detectable by the antibody. Use fresh samples with protease inhibitors and optimize lysis conditions.

• Non-specific binding: Secondary antibody binding to endogenous immunoglobulins or highly abundant proteins can create background. Include appropriate blocking conditions and consider using monoclonal antibodies for greater specificity .

For optimal results, researchers should compare band patterns across multiple MRPL9 antibodies targeting different epitopes and include appropriate controls.

How can I resolve weak or absent signals in MRPL9 immunofluorescence studies?

When troubleshooting weak MRPL9 immunofluorescence signals:

• Optimize fixation methods: Compare paraformaldehyde (4% PFA) with methanol fixation, as epitope accessibility may differ between methods .

• Adjust permeabilization conditions: Test different detergents (Triton X-100, saponin) and concentrations to improve antibody access to mitochondrial compartments.

• Implement signal amplification: Consider tyramide signal amplification or other signal enhancement methods for low-abundance proteins.

• Verify expression levels: Confirm MRPL9 expression in your cell line or tissue by Western blot before attempting immunofluorescence.

• Adjust antibody concentration: Increase primary antibody concentration (up to 1:50 dilution) for weak signals, ensuring appropriate controls for specificity .

• Extend incubation times: Consider overnight primary antibody incubation at 4°C to improve binding, particularly for tissue sections.

Published protocols demonstrating successful MRPL9 detection show clear mitochondrial staining patterns in MCF-7 cells using a 1:50 dilution of anti-MRPL9 antibody .

How can MRPL9 antibodies be used to explore the protein's role in cancer progression?

Based on recent findings about MRPL9's role in cancer, researchers can employ MRPL9 antibodies for:

The validated antibody applications (WB, IHC, IF) provide multiple approaches for these cancer research applications.

What methodological approaches should be used when studying the relationship between MRPL9 and mitochondrial function in cancer cells?

To investigate MRPL9's role in mitochondrial function in cancer:

• Subcellular fractionation and Western blotting:

  • Isolate mitochondrial, cytosolic, and nuclear fractions

  • Perform Western blotting with MRPL9 antibodies on each fraction

  • Include controls for fraction purity (e.g., VDAC for mitochondria, GAPDH for cytosol)

• Co-localization studies with mitochondrial markers:

  • Conduct dual immunofluorescence with MRPL9 antibodies and established mitochondrial markers (MitoTracker, TOMM20)

  • Quantify co-localization using appropriate software and statistical analysis

• Mitochondrial translation assessment:

  • Measure mitochondrial protein synthesis rates in MRPL9-depleted versus control cells

  • Use pulse-labeling techniques with radioactive amino acids to track translation

• Mitochondrial function analysis:

  • Assess oxygen consumption rate, ATP production, and mitochondrial membrane potential

  • Compare mitochondrial function parameters between MRPL9-normal and MRPL9-depleted cancer cells

• Structure-function relationship studies:

  • Use domain-specific antibodies to determine which regions of MRPL9 are critical for its function

  • Correlate structural features with functional outcomes in cancer models

These approaches leverage the specificity of MRPL9 antibodies to explore the connection between mitochondrial translation machinery and cancer phenotypes.

How should MRPL9 antibodies be validated for use in high-throughput screening applications?

For high-throughput screening applications involving MRPL9 antibodies:

• Batch-to-batch consistency validation:

  • Test multiple lots of the selected antibody against standard positive control samples

  • Quantify signal variation between batches using standardized Western blot or ELISA approaches

  • Establish acceptable performance thresholds for inter-batch variation

• Dynamic range assessment:

  • Determine the linear response range using recombinant MRPL9 protein standards

  • Establish detection limits (lower and upper) for the specific application platform

  • Create standard curves to enable semi-quantitative or quantitative analysis

• Automation compatibility testing:

  • Validate performance under automated liquid handling conditions

  • Assess stability under high-throughput storage and handling conditions

  • Optimize incubation times and washing steps for automated platforms

• Multiplex validation:

  • If used in multiplex assays, verify absence of cross-reactivity with other targets

  • Test for potential interference from common reagents in the screening platform

• Z-factor determination:

  • Calculate Z-factor using positive and negative controls to assess assay quality

  • Aim for Z-factor >0.5 for high-quality screening applications

These validation steps ensure reliable performance in high-throughput applications exploring MRPL9's role in cancer biology or mitochondrial function.

What considerations are important when designing MRPL9 antibody-based assays for detecting protein-protein interactions?

When designing assays to study MRPL9 protein interactions (such as with GGCT ):

• Epitope accessibility assessment:

  • Select antibodies with epitopes that don't interfere with known or predicted interaction domains

  • If studying the MRPL9-GGCT interaction, ensure the antibody binding site doesn't overlap with the interaction interface

• Co-IP protocol optimization:

  • Test different lysis buffer compositions to preserve native protein interactions

  • Optimize antibody-to-lysate ratios for maximum interaction capture

  • Compare direct and cross-linking approaches to capture transient interactions

• Proximity ligation assay (PLA) development:

  • For detecting in situ interactions, develop PLA protocols using MRPL9 antibodies paired with antibodies against suspected interaction partners

  • Validate specificity using known interactors (such as GGCT) and negative controls

• Pull-down verification approaches:

  • Complement antibody-based methods with tag-based approaches (e.g., FLAG-tagged MRPL9)

  • Verify interactions identified by antibody-based methods using orthogonal techniques

• Mass spectrometry compatibility:

  • If coupling to MS analysis, validate antibody performance under conditions compatible with downstream MS applications

  • Ensure the antibody can effectively immunoprecipitate sufficient protein for detection

Research has successfully used co-immunoprecipitation with anti-GGCT antibodies to demonstrate interaction with MRPL9, showing that compared to IgG controls, anti-GGCT enriched stronger MRPL9 signal .