MRPS36 Antibody

What is MRPS36 and why is it significant for mitochondrial research?

MRPS36 (Mitochondrial Ribosomal Protein S36) is a nuclear-encoded protein that plays a dual role in cellular biochemistry. While initially characterized as a component of the mitochondrial ribosome, recent research has established that MRPS36 is also a key structural member of the eukaryotic 2-oxoglutarate dehydrogenase complex (OGDHC) . Its significance stems from its exclusive presence in eukaryotes, where it provides a critical structural link in the OGDHC, mediating interactions between the E2o core and E3 components . This dual functionality makes MRPS36 an important target for researchers studying mitochondrial function, protein synthesis, and metabolic pathways, particularly the tricarboxylic acid (TCA) cycle.

What are the optimal applications for MRPS36 antibodies in research settings?

MRPS36 antibodies are versatile tools applicable across multiple experimental techniques. Based on available product information, these antibodies have been validated for:

• Western Blotting (WB): Useful for detecting MRPS36 protein expression levels and molecular weight confirmation

• Immunohistochemistry (IHC): Effective for localizing MRPS36 in tissue sections

• Enzyme-Linked Immunosorbent Assay (ELISA): Suitable for quantitative detection of MRPS36

• Immunocytochemistry (ICC): Applicable for cellular localization studies

When designing experiments, researchers should select antibodies specifically validated for their intended application. For optimized results in complex mitochondrial studies, combining methods such as Western blotting for expression level assessment with immunohistochemistry for localization analysis often provides more comprehensive insights into MRPS36 biology.

What is the species cross-reactivity profile of commercially available MRPS36 antibodies?

Commercial MRPS36 antibodies exhibit varying cross-reactivity profiles across species. Based on the search results, the antibody ABIN2791677 demonstrates broad cross-reactivity with multiple mammalian species, including:

• Human: 100% reactivity

• Mouse: 86% reactivity

• Rat: 93% reactivity

• Cow: 100% reactivity

• Horse: 93% reactivity

• Pig: 100% reactivity

Other antibodies, such as OACA03322, are primarily validated against human MRPS36 . This species variability is crucial to consider when designing experiments, particularly for comparative or evolutionary studies. Researchers should verify the predicted reactivity for their species of interest before antibody selection, as cross-reactivity is determined by sequence homology in the epitope region. For novel model organisms, preliminary validation may be necessary to confirm antibody functionality.

How should MRPS36 antibodies be stored and handled to maintain optimal activity?

For optimal preservation of MRPS36 antibody activity, follow these methodological guidelines:

• Storage temperature: Store antibodies at -20°C or -80°C for long-term preservation

• Formulation considerations: Commercial antibodies like OACA03322 are typically supplied in 50% glycerol and 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative

• Freeze-thaw cycles: Minimize repeated freeze-thaw cycles by aliquoting the antibody upon receipt

• Working dilutions: Prepare working dilutions fresh on the day of experiment and store at 4°C for short-term use

• Shipping considerations: Most antibodies are shipped with ice packs and remain stable for several days at ambient temperature

Following these handling protocols helps maintain epitope recognition capacity and ensures reproducible results across experiments. For specific antibody formulations that may differ from standard preparations, always consult the manufacturer's recommendations for optimal storage conditions.

What controls should be included when using MRPS36 antibodies in experimental designs?

Proper experimental controls are essential for validating results obtained with MRPS36 antibodies:

• Positive controls:

  • Tissues or cell lines with known MRPS36 expression (e.g., heart mitochondria, which show high MRPS36 expression)

  • Recombinant MRPS36 protein for antibody validation

• Negative controls:

  • Primary antibody omission to assess non-specific binding of secondary antibodies

  • Isotype controls (rabbit IgG for polyclonal antibodies) to evaluate background signal

  • MRPS36 knockdown or knockout samples when available

• Specificity controls:

  • Blocking peptide competition assays using the immunogenic peptide (e.g., the middle region peptide for ABIN2791677)

  • Western blot confirmation of a single band at the expected molecular weight

• Cross-validation:

  • Using multiple antibodies targeting different MRPS36 epitopes

  • Correlation of protein detection with mRNA expression data

These controls help distinguish specific from non-specific signals and validate antibody performance across different experimental conditions, ensuring reliable and reproducible results in MRPS36 research.

How can MRPS36 antibodies be leveraged to investigate its dual role in mitochondrial ribosomes and the oxoglutarate dehydrogenase complex?

To investigate MRPS36's dual functionality, researchers can implement the following methodological approaches:

• Subcellular fractionation combined with immunoprecipitation:

  • Isolate mitochondrial ribosomal fractions and OGDHC components separately

  • Use MRPS36 antibodies for immunoprecipitation from each fraction

  • Analyze co-precipitating proteins by mass spectrometry to identify differential interaction partners in each complex

• Proximity labeling approaches:

  • Generate MRPS36-BioID or APEX2 fusion constructs

  • Perform proximity labeling experiments in cellular systems

  • Compare biotinylated proteins between wildtype and cells with mutations in either ribosomal or OGDHC binding interfaces

  • Use MRPS36 antibodies to confirm expression and localization of fusion proteins

• Super-resolution microscopy:

  • Employ MRPS36 antibodies in combination with markers for mitochondrial ribosomes and OGDHC

  • Quantify co-localization coefficients under different metabolic conditions to assess dynamic association

• Cross-linking mass spectrometry approaches:

  • Following the methodology described in the literature, use chemical cross-linkers such as DSSO, PhoX, and DMTMM in intact mitochondria

  • Analyze MRPS36 cross-links to determine its interaction partners in both complexes

  • Validate findings with co-immunoprecipitation using MRPS36 antibodies

This multi-method approach provides complementary data on how MRPS36 distributes between and functions within these two critical mitochondrial complexes.

What are the key considerations when using MRPS36 antibodies for detecting post-translational modifications?

MRPS36 undergoes several post-translational modifications (PTMs), particularly phosphorylation, which may regulate its function. When investigating MRPS36 PTMs:

• Phosphorylation site-specific methodology:

  • Select antibodies that do not target regions containing known phosphorylation sites (e.g., S61)

  • For phosphorylation studies, consider phospho-specific antibodies if available

  • Use phosphatase treatments as controls to confirm phosphorylation-dependent signals

• Sample preparation considerations:

  • Include phosphatase inhibitors during tissue/cell lysis

  • For MS-based approaches, enrich phosphopeptides using titanium dioxide or immobilized metal affinity chromatography

  • Consider native conditions to preserve physiological modification states

• Detection strategy:

  • Use Phos-tag gels for mobility shift detection of phosphorylated MRPS36

  • Implement 2D gel electrophoresis to separate differentially modified MRPS36 isoforms

  • Follow with Western blotting using MRPS36 antibodies

• Validation approach:

  • Correlate antibody results with mass spectrometry data

  • Use site-directed mutagenesis of key residues (e.g., S61 to A) to confirm specificity

  • Compare modification patterns across different physiological and stress conditions

As reported in the literature, MRPS36 has confirmed phosphorylation sites, such as S61, which may regulate its interaction with E2o and E3 components of the OGDHC complex .

How can researchers troubleshoot inconsistent results when using MRPS36 antibodies in cross-linking mass spectrometry studies?

Cross-linking mass spectrometry (XL-MS) is a powerful technique for studying MRPS36 interactions, but can present technical challenges. When troubleshooting inconsistent results:

• Cross-linker optimization:

  • Test multiple cross-linkers with different spacer lengths (e.g., DSSO, PhoX, DMTMM as described in the literature)

  • Optimize cross-linker concentration and reaction time for specific sample types

  • Consider membrane-permeable vs. impermeable cross-linkers depending on sample preparation

• Sample preparation refinement:

  • Ensure mitochondrial integrity by checking respiratory control ratios

  • Use fresh samples when possible to minimize protein degradation

  • Confirm MRPS36 antibody epitope accessibility after cross-linking via Western blot

• Data analysis strategy:

  • Implement stringent filtering criteria for cross-link identification

  • Validate interactions through multiple biological and technical replicates

  • Compare results across different organisms (e.g., bovine and mouse heart mitochondria) to identify conserved interactions

• Validation approach:

  • Confirm key cross-links with alternative methods (e.g., co-immunoprecipitation)

  • Perform competition assays with peptides corresponding to the predicted interaction sites

  • Generate structural models incorporating cross-linking distance constraints

For optimal results, researchers should consider using multiple cross-linkers and complementary approaches such as complex profiling (CP) to corroborate XL-MS findings, as demonstrated in recent MRPS36 studies .

What are the implications of MRPS36's evolutionary profile for antibody selection in comparative studies?

The evolutionary profile of MRPS36 has significant implications for antibody selection in cross-species research:

• Evolutionary constraints:

  • MRPS36 homologs are exclusively found in eukaryotes, with no prokaryotic counterparts

  • Within eukaryotes, there is sequence divergence that may affect epitope conservation

  • The N-terminal and C-terminal regions show different conservation patterns, with functional constraints on regions that interact with E2o and E3

• Epitope selection strategy:

  • Target highly conserved regions for broad cross-reactivity across species

  • The middle region targeted by some commercial antibodies (e.g., ABIN2791677) shows good conservation across mammals

  • For species-specific studies, select antibodies against divergent regions

• Validation requirements:

  • Perform sequence alignment of the immunogen sequence across target species

  • Validate antibody performance in each species through Western blotting

  • Consider raising custom antibodies against species-specific sequences for divergent organisms

• Functional domain considerations:

  • Select antibodies that recognize functionally relevant domains based on research questions

  • For studying E3 interactions, target N-terminal epitopes

  • For E2o interactions, C-terminal-targeting antibodies may be more informative

This evolutionary context is particularly important when designing comparative studies across distantly related species, as epitope conservation directly impacts antibody performance.

How can researchers design experiments to investigate the role of MRPS36 in the structural organization of the 2-oxoglutarate dehydrogenase complex?

To investigate MRPS36's structural role in the OGDHC, researchers can implement these experimental approaches:

• Structure-function analysis:

  • Use domain-specific MRPS36 antibodies to block specific interaction interfaces

  • Assess the impact on OGDHC assembly and activity in reconstituted systems

  • Perform site-directed mutagenesis of key residues identified in cross-linking studies

  • Correlate structural perturbations with enzymatic activity measurements

• Cryo-EM methodology:

  • Purify intact OGDHC complexes using gentle conditions that preserve MRPS36 associations

  • Perform immunogold labeling with MRPS36 antibodies to localize it within the complex

  • Compare structures of OGDHC with and without MRPS36 to determine its structural contribution

  • Correlate findings with cross-linking data to build comprehensive structural models

• In vivo knockdown studies:

  • Generate MRPS36-depleted cell lines using RNAi or CRISPR-Cas9

  • Assess OGDHC assembly using BN-PAGE followed by Western blotting with MRPS36 antibodies

  • Measure OGDHC activity and TCA cycle flux in knockout versus control cells

  • Rescue experiments with wildtype versus mutant MRPS36 to validate functional domains

• Protein-protein interaction mapping:

  • Use MRPS36 antibodies to perform co-immunoprecipitation under various conditions

  • Implement hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Correlate with current models suggesting MRPS36 mediates interactions between E2o and E3

These approaches collectively provide insights into how MRPS36 contributes to the eukaryotic OGDHC architecture, estimated to be approximately 3.45 MDa with 24 E2o subunits, 16 E1o subunits, 12 E3 subunits, and 6 MRPS36 subunits .

Table 1: Comparative Analysis of MRPS36 Antibody Specifications and Research Applications

Table 2: MRPS36 Function in Experimental Systems and Antibody Application Context