MRPL27 Antibody

What is MRPL27 and why is it significant for mitochondrial research?

MRPL27 (Mitochondrial Ribosomal Protein L27) is a component of the large 39S subunit of mitochondrial ribosomes. These mitoribosomes are essential for protein synthesis within mitochondria. Unlike prokaryotic ribosomes, mammalian mitoribosomes have an estimated 75% protein to rRNA composition (a reversed ratio compared to prokaryotic ribosomes) . MRPL27 is encoded by nuclear genes but functions within mitochondria, making it an important marker for studying mitochondrial translation processes . The protein has a calculated molecular weight of 16 kDa but is typically observed at approximately 10 kDa in experimental conditions .

What validated applications exist for MRPL27 antibodies in research?

MRPL27 antibodies have been validated for multiple experimental applications:

Positive detection has been confirmed in several cell types, including A431 cells for IF/ICC applications .

What reactivity profile do MRPL27 antibodies demonstrate across species?

Most commercially available MRPL27 antibodies demonstrate cross-reactivity across multiple mammalian species:

This cross-reactivity makes these antibodies valuable for comparative studies across different model organisms in mitochondrial research.

What are the optimal storage conditions for maintaining MRPL27 antibody stability?

For maximum stability and reactivity maintenance, MRPL27 antibodies should be:

• Stored at -20°C in aliquots to prevent repeated freeze-thaw cycles

• Maintained in storage buffer consisting of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Expected shelf life is typically one year after shipment when stored properly

• For smaller quantities (20μl sizes), products may contain 0.1% BSA for additional stability

Research indicates that aliquoting is generally unnecessary for -20°C storage, but it becomes important if frequent access is required .

What experimental controls should be implemented when using MRPL27 antibodies?

Proper controls are essential for ensuring specificity and reliability:

• Positive tissue controls: Mouse or human liver tissue has been successfully used for WB applications

• Positive cell line controls: A431 cells for IF/ICC applications

• Negative controls: Use of secondary antibody alone and/or isotype controls

• Knockdown validation: Where possible, validate specificity using MRPL27 knockdown or knockout samples

• Antigen blocking: Pre-incubation with immunizing peptide can confirm specificity

Each antibody should be titrated in the specific testing system to obtain optimal results, as performance may be sample-dependent .

How should sample preparation be optimized for detecting MRPL27 in different applications?

Sample preparation varies by application:

For Western Blot:

• Use standard RIPA or NP-40 based lysis buffers with protease inhibitors

• Include mitochondrial enrichment steps for enhanced detection

• Expected molecular weight is 10 kDa (observed) versus 16 kDa (calculated)

For IHC applications:

• Antigen retrieval with TE buffer pH 9.0 is recommended

• Alternative antigen retrieval may be performed with citrate buffer pH 6.0

• Dilution ranges of 1:20-1:200 are suggested

For IF/ICC applications:

• Fixation with 4% paraformaldehyde followed by permeabilization

• Optimal dilution range of 1:200-1:800

How can MRPL27 antibodies be used to study mitochondrial translation defects?

MRPL27 antibodies provide valuable tools for investigating mitochondrial translation defects through several approaches:

Methodologically, these studies typically combine immunoblotting with functional assays of mitochondrial translation (e.g., 35S-methionine labeling).

What is known about MRPL27's role in disease pathogenesis?

Current research suggests MRPL27 may play roles in disease contexts:

• Myasthenia Gravis association: A study demonstrated that miR-1933-3p is upregulated in MuSK+ Experimental Autoimmune Myasthenia Gravis (EAMG), leading to reduced expression of MRPL27. This reduction may contribute to mitochondrial dysfunction and muscle atrophy observed in the disease .

• Potential mitochondrial disease link: By analogy with other mitoribosomal proteins like MRPS25, whose mutations cause human disease, MRPL27 dysfunction might similarly impact mitochondrial translation and consequently oxidative phosphorylation .

• Methodological approach: Detection of MRPL27 expression changes in disease models requires careful normalization and comparison across affected and control tissues, with particular attention to cell-type specific expression patterns.

How do MRPL27 antibodies compare with antibodies against other mitoribosomal proteins for studying mitochondrial translation?

Comparative analysis of mitoribosomal protein antibodies reveals distinct advantages:

When studying mitochondrial versus cytoplasmic translation, the combined use of MRPL27 and RPL27 antibodies provides a methodological advantage by distinguishing between these separate translation systems .

What technical challenges exist in detecting MRPL27 in experimental systems?

Several technical challenges require consideration:

• Small protein size: With an observed molecular weight of just 10 kDa, MRPL27 requires special attention to gel percentage and running conditions in Western blot applications .

• Mitochondrial localization: Since MRPL27 is localized to mitochondria, subcellular fractionation may be necessary for enrichment in samples with low mitochondrial content.

• Cross-reactivity concerns: Researchers should be aware of potential cross-reactivity with RPL27 (cytoplasmic ribosomal protein L27) , which necessitates careful antibody selection and validation.

• Detection sensitivity: The recommended dilutions for MRPL27 antibodies (1:200-1:800 for IF/ICC and 1:500-1:2000 for WB) suggest moderate abundance, requiring optimization of detection methods .

How can MRPL27 antibodies contribute to understanding mitochondrial translation regulation?

MRPL27 antibodies are instrumental in several emerging research areas:

• Mitoribosome structural studies: Co-immunoprecipitation using MRPL27 antibodies can help identify interaction partners and structural components, complementing cryo-EM approaches.

• Translational adaptation: Monitoring MRPL27 levels under different cellular stresses can reveal how mitochondrial translation adapts to changing energy demands.

• Mitochondrial-nuclear communication: MRPL27 antibodies can be used to study how nuclear-encoded mitoribosomal components respond to mitochondrial signaling, potentially through techniques like proximity labeling.

The ideal methodological approach combines quantitative immunoblotting with functional assessment of mitochondrial translation and energy production .

What complementary approaches should be used alongside MRPL27 antibodies for comprehensive mitoribosomal analysis?

A multi-faceted approach yields the most complete understanding:

• Combined antibody panels: Using antibodies against multiple mitoribosomal components (both 28S and 39S subunits) provides insights into global versus specific effects on the mitoribosome .

• Functional translation assays: Coupling MRPL27 antibody detection with 35S-methionine labeling of newly synthesized mitochondrial proteins.

• mRNA and rRNA analysis: Quantifying 12S and 16S rRNA levels alongside MRPL27 protein detection offers insights into coordination between mitoribosomal proteins and RNA components .

• Sucrose gradient analysis: Using MRPL27 antibodies to track mitoribosome assembly by analyzing the distribution in sucrose gradient fractions has proven valuable in mitoribosomal pathology research .

• Genetic complementation: In cells with suspected mitochondrial translation defects, restoring wild-type expression can confirm functional roles .

This integrated approach has successfully elucidated mitoribosomal dysfunction in conditions like MRPS25 deficiency and holds promise for understanding MRPL27's role.

What strategies can address non-specific binding when using MRPL27 antibodies?

Non-specific binding can be mitigated through several methodological approaches:

• Optimization of blocking: Use 5% non-fat milk or BSA in TBS-T for Western blots, with extended blocking times of 1-2 hours at room temperature.

• Antibody titration: Following manufacturer recommendations for dilution ranges (WB: 1:500-1:2000; IF/ICC: 1:200-1:800; IHC: 1:20-1:200) but systematically testing within these ranges for specific samples .

• Increased washing: Implementing additional washing steps with 0.1-0.3% Tween-20 in PBS or TBS.

• Secondary antibody controls: Include controls with secondary antibody alone to detect potential non-specific binding from this source.

• Peptide competition: Pre-incubation of the antibody with the immunizing peptide can confirm specificity of the observed signal.

Most commercial MRPL27 antibodies undergo affinity purification, which helps reduce non-specific binding .

How should researchers validate MRPL27 antibody specificity for their particular experimental system?

Rigorous validation requires multiple complementary approaches:

• Western blot validation: Use known positive samples such as human or mouse liver tissue to confirm detection at the expected size (10 kDa observed, 16 kDa calculated) .

• Knockdown/knockout validation: Compare antibody signal between wild-type and MRPL27-depleted samples.

• Cellular localization: Confirm mitochondrial localization pattern in IF/ICC applications, potentially through co-staining with established mitochondrial markers.

• Cross-reactivity assessment: Test for potential cross-reactivity with RPL27 (cytoplasmic ribosomal protein L27) , especially when working with whole cell lysates.

• Multiple antibody comparison: When possible, compare results obtained with antibodies from different manufacturers or those targeting different epitopes.

Each experimental system may require specific optimization of these validation approaches.