MRPL19 Antibody

What is MRPL19 and why is it significant in research?

MRPL19 (Mitochondrial Ribosomal Protein L19) is a nuclear-encoded protein that functions in mitochondrial protein synthesis. It is a component of the 39S large subunit of mitochondrial ribosomes (mitoribosomes). Unlike prokaryotic ribosomes, mitoribosomes have an estimated 75% protein to rRNA composition, making proteins like MRPL19 particularly important for their function . MRPL19 has gained research significance due to its association with cancer progression, particularly in lung adenocarcinoma where it has been linked to lymph node metastasis, differentiation level, and tumor status .

What are the primary applications for MRPL19 antibodies in research?

MRPL19 antibodies are versatile research tools with multiple validated applications:

How can researchers validate the specificity of MRPL19 antibodies?

Validation is critical for ensuring experimental reliability:

• Positive control selection: Use tissues/cell lines known to express MRPL19, such as HeLa cells, 293T, A431, Jurkat, or Raji cells .

• Molecular weight verification: Confirm detection at the expected 34 kDa molecular weight via Western blot .

• Knockdown validation: Compare antibody signal in control vs. MRPL19 knockdown samples. RNA interference using shRNA against MRPL19 (as used in A549 and H1299 cell lines) provides a reliable negative control .

• Cross-reactivity assessment: Test antibody against related mitochondrial ribosomal proteins to ensure specificity.

What sample preparation methods are optimal for MRPL19 detection?

Sample preparation significantly impacts MRPL19 detection:

For Immunohistochemistry:

• Fix tissues in 4% paraformaldehyde

• For paraffin-embedded sections, optimal antigen retrieval uses TE buffer pH 9.0, though citrate buffer pH 6.0 can serve as an alternative

• Counterstain with hematoxylin for visualization

For Western Blotting:

• Extract proteins using RIPA buffer with protease inhibitors

• Confirm protein concentration using Bradford or BCA assay

• Load 20-40 μg protein per lane

• Use proper positive controls (e.g., HeLa cells lysate)

For Immunofluorescence:

• Fix cells with 4% paraformaldehyde (10-15 minutes)

• Permeabilize with 0.1-0.5% Triton X-100

• Block with 1-5% BSA or normal serum

• Use MRPL19 antibody at 1:50-1:500 dilution

How can MRPL19 antibodies be used to investigate cancer progression?

MRPL19 antibodies offer powerful tools for cancer research:

• Clinical correlation studies: Use IHC to quantify MRPL19 expression in patient tumor samples and correlate with clinicopathological features. Scoring can be calculated by multiplying staining intensity (0-3) by stained regions percentage (0-4) .

• Prognostic analysis: Using Kaplan-Meier survival analysis, researchers can correlate MRPL19 expression levels with patient outcomes. High MRPL19 expression has been associated with poor prognosis in LUAD .

• Functional assessment: After MRPL19 knockdown (using siRNA or shRNA), utilize antibodies to confirm protein reduction and examine effects on:

  • Cell proliferation

  • Migration and invasion capabilities

  • Signaling pathway activation

• Immune infiltration analysis: Combine MRPL19 staining with immune cell markers to investigate correlations between MRPL19 expression and immune cell infiltration. Studies have shown relationships between MRPL19 expression and B cells, CD4+ T cells, and dendritic cell infiltration in LUAD .

What methodologies effectively evaluate MRPL19's role in mitochondrial function?

To investigate MRPL19's role in mitochondrial biology:

• Co-localization studies: Perform dual immunofluorescence with MRPL19 antibody and mitochondrial markers (e.g., MitoTracker or TOMM20) to confirm mitochondrial localization.

• Mitochondrial ribosome profiling: Use MRPL19 antibodies for immunoprecipitation of mitoribosome complexes followed by RNA-seq to identify associated transcripts.

• Mitochondrial translation assays: Following MRPL19 knockdown, measure translation of mitochondrially-encoded proteins using metabolic labeling with 35S-methionine in the presence of cytoplasmic translation inhibitors.

• Respiratory chain analysis: Correlate MRPL19 levels with mitochondrial respiration parameters using oxygen consumption rate measurements.

How do researchers address contradictory MRPL19 expression data across different experimental systems?

When facing contradictory results:

• Antibody validation comparison: Different antibody clones may recognize distinct epitopes. Compare results using antibodies targeting different regions of MRPL19 (e.g., N-terminal vs. C-terminal epitopes) .

• Isoform consideration: Verify which MRPL19 isoform your antibody detects, as alternative splicing may occur.

• Normalization approach: Ensure proper normalization in expression studies:

  • For Western blots: Use appropriate loading controls (β-actin, GAPDH)

  • For IHC: Implement standardized scoring systems and blinded pathologist evaluation

• Cell line heterogeneity: Different cell lines show variable MRPL19 expression. NCI-H1299 and A549 cell lines demonstrate higher MRPL19 levels than 95-D cells .

What considerations are important for multiplexed detection involving MRPL19?

For multiplexed detection:

• Antibody compatibility: When co-staining, select MRPL19 antibodies from different host species than your other target antibodies (e.g., rabbit anti-MRPL19 with mouse anti-CD4).

• Fluorophore selection: Choose fluorophores with minimal spectral overlap:

  • FITC-conjugated MRPL19 antibodies (Ex/Em: 495nm/519nm) work well with red and far-red fluorophores

  • Consider using Zenon labeling technology for same-species antibodies

• Sequential staining protocol:

  • Perform heat-mediated antigen retrieval once

  • Apply first primary antibody, wash, then appropriate secondary

  • Block with excess normal IgG from first antibody species

  • Apply second primary and secondary antibodies

  • Include appropriate controls for each antibody individually

How can researchers utilize MRPL19 antibodies to study its role in immune response modulation?

Building on MRPL19's connection to immune infiltration:

• Spatial analysis: Use multiplex immunofluorescence with MRPL19 and immune cell markers to analyze spatial relationships between MRPL19-expressing cancer cells and infiltrating immune cells.

• TIMER database integration: Combine MRPL19 IHC data with computational approaches using the TIMER database to assess relationships between MRPL19 expression, tumor purity, and infiltrating immune cells .

• Copy number variation effects: Investigate how MRPL19 copy number alterations affect immune cell infiltration using the SCNA module of TIMER .

• T-helper cell differentiation: Since MRPL19 may be associated with T-helper cell differentiation, design co-culture experiments with MRPL19-manipulated cancer cells and T cells to evaluate differentiation effects .

How should researchers address weak or absent MRPL19 signal in Western blots?

For optimal Western blot detection:

• Extraction optimization: Ensure complete protein extraction using buffers containing 1% Triton X-100 or RIPA buffer with protease inhibitors.

• Loading amount: Increase protein loading to 40-60 μg per lane when detecting endogenous MRPL19.

• Transfer efficiency: Use semi-dry transfer at 15V for 30-45 minutes or wet transfer at 30V overnight at 4°C.

• Blocking optimization: Test 5% non-fat milk vs. 3-5% BSA in TBS-T for blocking.

• Primary antibody incubation: Extend incubation to overnight at 4°C using 1:500 dilution .

• Enhanced detection: Utilize high-sensitivity ECL substrates or consider signal amplification systems.

What strategies help overcome background issues in MRPL19 immunostaining?

To reduce background and improve signal-to-noise ratio:

• Antigen retrieval optimization: Compare heat-mediated retrieval with TE buffer pH 9.0 vs. citrate buffer pH 6.0 .

• Blocking enhancement:

  • Extend blocking to 1-2 hours at room temperature

  • Use species-specific serum that matches your secondary antibody

  • Add 0.1-0.3% Triton X-100 to blocking solution for permeabilization

• Antibody dilution series: Test serial dilutions (1:200, 1:400, 1:800) to determine optimal concentration .

• Washing protocol: Implement stringent washing (5 × 5 minutes in TBS-T) after primary and secondary antibody incubations.

• Fluorescence considerations: For IF applications, include an extra quenching step (0.1% sodium borohydride) to reduce autofluorescence.

How might MRPL19 antibodies contribute to understanding mitochondrial dysfunction in cancer?

Emerging research avenues include:

• Metabolic reprogramming: Investigate correlations between MRPL19 expression and metabolic markers in cancer tissues.

• Mitochondrial stress response: Study MRPL19 expression changes during mitochondrial stress using antibodies to track protein levels and localization.

• Therapeutic targeting assessment: Use MRPL19 antibodies to evaluate the effects of mitochondrial-targeted therapies on mitoribosome integrity.

• Cancer stem cell connection: Explore potential roles of MRPL19 in cancer stem cell maintenance by co-staining with stemness markers.

• Drug resistance mechanisms: Investigate whether MRPL19 overexpression contributes to chemoresistance by comparing expression in sensitive versus resistant cell populations.

What methodological approaches can advance MRPL19 research beyond lung adenocarcinoma?

To expand MRPL19 research:

• Multi-cancer tissue microarrays: Apply validated MRPL19 IHC protocols to tissue microarrays spanning multiple cancer types.

• Single-cell analysis: Combine MRPL19 antibodies with single-cell technologies to examine expression heterogeneity within tumors.

• Patient-derived organoids: Use MRPL19 immunostaining to characterize expression in 3D organoid models from various cancer types.

• Liquid biopsy integration: Explore correlations between tissue MRPL19 expression and circulating tumor cell characteristics.

• Combination biomarker approach: Investigate MRPL19 in combination with other mitochondrial ribosomal proteins to develop more robust prognostic signatures.