MRPL44 Antibody

What is MRPL44 and why is it important in cellular research?

MRPL44 (mitochondrial ribosomal protein L44) is a 332 amino acid protein with a molecular weight of approximately 38 kDa that localizes to the mitochondrial matrix. It functions as a component of the 39S subunit of mitochondrial ribosomes . MRPL44 plays crucial roles in:

• Regulation of mtDNA-encoded gene expression at both RNA and protein levels

• Mitochondrial protein translation

• Maintenance of oxidative phosphorylation (OXPHOS) capacity

• ATP synthesis dependent on Complex I and Complex II

• Mitochondrial respiratory capacity

Research has demonstrated that knockdown of MRPL44 results in decreased levels of mtDNA-encoded RNA (particularly 16S rRNA, ND2, ND4, and ND5) and reduced mitochondrial protein translation, ultimately impairing ATP synthesis and respiratory capacity .

Which applications are most commonly validated for MRPL44 antibodies?

MRPL44 antibodies have been extensively validated for multiple applications:

For optimal results, it is recommended to titrate the antibody concentration for each specific experimental system and sample type . Validation data typically includes positive detection in multiple cell lines including A549, HeLa, Jurkat, K-562, and human tissue samples .

How should researchers select the appropriate MRPL44 antibody for their specific application?

Selection of the optimal MRPL44 antibody should be based on:

• Target species compatibility: Verify reactivity with your experimental model. Most commercial MRPL44 antibodies are reactive with human, mouse, and rat samples . Some also show reactivity with monkey, zebrafish, and yeast models .

• Application requirements:

  • For subcellular localization studies, choose antibodies validated for immunofluorescence microscopy

  • For protein expression analysis, select antibodies with strong Western blot validation

  • For protein-protein interaction studies, use antibodies validated for immunoprecipitation

• Immunogen consideration: Different antibodies target different epitopes:

  • Some target full-length MRPL44 protein

  • Others target specific peptide sequences (e.g., Sigma's HPA038147 targets amino acids GLLVEELKKRNVSAPESRLTRQSGGTTALPLYFVGLYCDKKLIAEGPGETVLVAEEEAARVALRKLYGFTENRRPWNYSKPKETLRAE)

  • Some target internal regions of human MRPL44

• Validation extent: Review available validation data including:

  • Western blot band specificity at expected 38 kDa

  • Cross-reactivity testing against multiple proteins

  • Published application examples

What are the recommended protocols for subcellular fractionation to study MRPL44 localization?

For accurate investigation of MRPL44 subcellular localization, researchers should use the following validated protocols:

• Mitochondrial isolation:

  • Fractionate cells into nuclei, cytosol (± heavy membranes), mitochondria, and mitochondrial components

  • Extract mitochondrial fraction with digitonin as described in published protocols

  • Verify fraction purity using established markers (e.g., mitochondrial markers, cytosolic markers)

• Mitoplast preparation:

  • Separate mitochondrial outer membrane and intermembrane space (IO) from the mitoplast (MP, mitochondrial inner membrane plus matrix)

  • Analysis of endogenous MRPL44 by Western blotting has confirmed predominant localization to the MP fraction

• Immunofluorescence microscopy:

  • Grow cells on gelatin-coated coverslips

  • Stain mitochondria with MitoTrackerRed 30 min prior to harvesting

  • Fix cells with paraformaldehyde

  • Use appropriate MRPL44 antibody (1:20-1:200 dilution range is typical)

  • Include DAPI for nuclear visualization

  • Image using fluorescence microscopy (widefield or confocal)

Co-localization studies have confirmed that MRPL44 specifically localizes to mitochondria but not to endoplasmic reticulum or actin filaments .

How can researchers effectively study MRPL44's role in mitochondrial translation?

To investigate MRPL44's function in mitochondrial translation:

• Mitochondrial translation assay:

  • Label mitochondrial translation products with 35S-methionine

  • Use emetine to inhibit cytosolic translation

  • Follow a time course (0-60 min) to assess translation rates

  • Analyze labeled products by SDS-PAGE and autoradiography

• Gene knockdown approach:

  • Target MRPL44 using validated shRNAs (e.g., targeting 3' UTR sequence 5'-TCTCTTACACACTGGTTTATTACT-3' or ORF sequence 5'-GGAAAGAGCTCTTTGAGATGT-3')

  • Confirm knockdown efficiency by Western blot

  • Assess effects on mitochondrial protein synthesis

Research has demonstrated that MRPL44 knockdown results in:

• Gene overexpression studies:

  • Express tagged versions (FLAG or GFP) of MRPL44

  • Assess effects on mitochondrial RNA levels and protein expression

  • Investigate impacts on mitochondrial function

What approaches can be used to investigate MRPL44's protein-protein interactions within the mitoribosome?

For comprehensive characterization of MRPL44's interactions:

• Co-immunoprecipitation (Co-IP):

  • Express tagged MRPL44 (e.g., FLAG-tagged or GFP-tagged)

  • Isolate heavy membrane fractions using digitonin permeabilization

  • Lyse in appropriate buffer (e.g., KALB lysis buffer: 20mM Tris-HCl pH 8.0, 100mM KCl, 0.2 mM EDTA)

  • Immunoprecipitate using anti-tag antibodies or anti-MRPL44 antibodies

  • Analyze bound proteins by SDS-PAGE and Western blotting

• Protein-RNA interaction analysis:

  • Capture MRPL44 with antibodies

  • Extract bound RNA

  • Analyze by quantitative RT-PCR for mitochondrial rRNAs (12S and 16S)

  • Results have confirmed that both 12S and 16S rRNAs co-immunoprecipitate with MRPL44

• Size exclusion chromatography:

  • Perform HPLC on cell extracts

  • Analyze elution profiles of MRPL44 and other mitoribosomal proteins

  • Research shows MRPL44 elutes primarily in complexes >670 kDa and some smaller complexes <158 kDa

  • The larger complex co-elutes with known mitoribosomal components (Mrpl12 and Mrps15)

What are the most common pitfalls in MRPL44 antibody-based experiments and how can they be addressed?

Common challenges and solutions include:

• Nonspecific binding in Western blot:

  • Optimize antibody dilution (1:5000-1:50000 recommended for WB)

  • Increase blocking time/concentration

  • Use validated positive controls (A549, HeLa, Jurkat, K-562 cells have confirmed expression)

  • Include appropriate negative controls (knockdown samples)

  • Verify expected molecular weight (38 kDa)

• Weak or absent signal in immunohistochemistry:

  • Optimize antigen retrieval (use TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Adjust antibody concentration (1:20-1:200 is typically effective)

  • Extend primary antibody incubation time

  • Use more sensitive detection systems

  • Verify tissue expression patterns with literature

• Inconsistent results across experiments:

  • Store antibody properly (-20°C, avoid repeated freeze-thaw cycles)

  • Prepare consistent lysates (use standardized protocols)

  • Include positive controls in each experiment

  • Standardize sample preparation and loading

• Cross-reactivity concerns:

  • Validate antibody specificity using knockdown controls

  • Confirm single band at expected molecular weight

  • Consider using antibodies verified on protein arrays against 383+ non-specific proteins

How can researchers effectively analyze MRPL44's impact on mitochondrial function?

To comprehensively assess MRPL44's functional roles:

• ATP synthesis measurement:

  • Permeabilize cells with digitonin

  • Measure ATP synthesis under different substrate conditions:

    • Complex I-dependent: glutamate and malate (± rotenone)

    • Complex II-dependent: succinate with rotenone (± malonate)

  • Normalize rates to control Complex II-dependent rate

  • MRPL44 knockdown has been shown to reduce both Complex I and Complex II-dependent ATP synthesis rates

• Oxygen consumption rate (OCR) analysis:

  • Measure parameters including:

    • Resting OCR

    • Maximal respiratory rate (MRR)

    • Spare respiratory capacity (SRC)

  • Monitor extracellular acidification rate (ECAR) as an indirect measure of glycolytic activity

  • Calculate OCR/ECAR ratio to determine cellular preference for OXPHOS vs. glycolysis

  • Research shows MRPL44 knockdown reduces OCR, MRR and SRC, while overexpression increases these parameters

• OXPHOS complex assembly analysis:

  • Assess formation of respiratory chain complexes

  • MRPL44 knockdown has been shown to reduce assembly of Complexes I, III, IV and V

What are the emerging research areas regarding MRPL44's potential role in disease states?

Recent findings highlight several promising research directions:

• Mitochondrial infantile cardiomyopathy:

  • MRPL44 mutation (p.L156R) within the RNase III domain has been associated with this condition

  • This mutation may affect protein structure rather than RNA binding

  • Research should focus on identifying other pathogenic mutations that may contribute to mitochondrial syndromes

• Cancer metabolism:

  • Investigate MRPL44 expression in various cancer types

  • MRPL44 antibodies have been validated in human ovary tumor tissue

  • Research potential correlations between MRPL44 expression/function and cancer cell metabolism

• RNase III domain functionality:

  • Although bioinformatics analysis suggests critical residues for RNase activity are missing in mammalian MRPL44

  • Further investigation into potential RNA processing functions is warranted

  • Analysis of mitochondrial-wide RNA processing in MRPL44 mutant cells could be informative

• Therapeutic targeting:

  • Explore potential for therapeutic modulation of MRPL44 in mitochondrial diseases

  • Investigate small molecule approaches to enhance MRPL44 function in disease states

How can researchers integrate MRPL44 studies with broader mitochondrial ribosome research?

For comprehensive integration:

• Structural biology approaches:

  • Use cryo-EM to determine MRPL44's precise position within the mitoribosome

  • Compare structural features across species to understand evolutionary conservation

  • Investigate potential conformational changes during translation

• System-wide analysis:

  • Combine MRPL44 studies with analysis of other mitoribosomal proteins

  • Perform proteomics of isolated mitoribosomes under different conditions

  • Investigate coordination between nuclear and mitochondrial translation systems

• Translational regulation studies:

  • Investigate how MRPL44 contributes to selective translation of mitochondrial mRNAs

  • Study the integration of newly synthesized proteins into OXPHOS complexes

  • Analyze the interplay between mitochondrial translation and import of nuclear-encoded mitochondrial proteins

• Evolutionary perspectives:

  • Compare MRPL44 structure and function across species

  • Current antibodies show reactivity with human, mouse, rat, monkey, zebrafish, and yeast

  • Explore functional conservation and divergence of MRPL44 across evolutionary distance