MRPS28 Antibody

What is MRPS28 and why is it important in research?

MRPS28, also known as 28S ribosomal protein S28 or mitochondrial ribosomal protein S28, is a nuclear-encoded protein that functions in the mitochondrial ribosome. It plays a crucial role in mitochondrial protein synthesis and is a component of the small mitochondrial ribosomal subunit (28S). MRPS28 is important in research related to mitochondrial function, energy metabolism, and diseases associated with mitochondrial dysfunction .

The protein has several aliases including 28S ribosomal protein S35 (mitochondrial), MRP-S28, MRP-S35, MRPS35, HSPC007, S28mt, and S35mt . Its calculated molecular weight is approximately 20.8 kDa . MRPS28 belongs to the bacterial ribosomal protein bS1 family and is classified as a mitochondrial small ribosomal subunit protein (bS1m) .

What types of MRPS28 antibodies are available for research?

Several types of MRPS28 antibodies are available for research purposes:

• Host species variations:

  • Rabbit polyclonal antibodies (e.g., Boster Bio A13408)

  • Mouse polyclonal antibodies (e.g., Novus Biologicals H00028957-B01P)

• Reactivity profiles:

  • Human-specific antibodies

  • Mouse-reactive antibodies

  • Multi-species reactive antibodies (e.g., human and mouse)

• Application-optimized antibodies:

  • Western Blot (WB) validated antibodies

  • Immunocytochemistry/Immunofluorescence (ICC/IF) validated antibodies

When selecting an antibody, researchers should consider the specific experimental requirements, including target species, application type, and validation data available for the antibody .

What are the recommended storage and handling conditions for MRPS28 antibodies?

Proper storage and handling of MRPS28 antibodies are essential for maintaining their activity and specificity. Based on manufacturer recommendations:

Important handling considerations include:

• Avoid repeated freeze-thaw cycles as they can degrade antibody quality and reduce specificity .

• Most MRPS28 antibodies are supplied in a stabilizing buffer (e.g., PBS with 0.02% sodium azide, 50% glycerol, pH 7.2) that helps maintain antibody integrity .

• When working with aliquots, it's recommended to store them at -20°C or -80°C to preserve antibody function over time .

• Some formulations may contain preservatives like sodium azide, which is toxic and should be handled with appropriate safety precautions .

Proper adherence to these storage recommendations will help ensure optimal antibody performance in experimental applications.

What are the basic applications for MRPS28 antibodies?

MRPS28 antibodies have been validated for several standard laboratory applications, including:

• Western Blotting (WB): Used to detect and quantify MRPS28 protein in cell or tissue lysates. Recommended dilutions typically range from 1:500 to 1:1000 .

• Immunocytochemistry (ICC): Used to visualize the cellular localization of MRPS28 in cultured cells. Recommended dilutions are typically between 1:20 and 1:100 .

• Immunofluorescence (IF): Similar to ICC but specifically using fluorescent detection methods to visualize protein localization. Recommended dilutions are typically between 1:20 and 1:100 .

These applications allow researchers to study MRPS28 expression levels, subcellular localization, and potential alterations in different experimental conditions or disease states .

How should experimental design be optimized when using MRPS28 antibodies?

When designing experiments using MRPS28 antibodies, several critical factors should be considered for optimal results:

• Experimental controls:

  • Positive controls: Include samples known to express MRPS28 (e.g., cell lines with confirmed expression)

  • Negative controls: Include samples without the primary antibody to establish background signal levels

  • Loading controls: For western blots, include housekeeping proteins (e.g., β-actin, GAPDH) for normalization

  • Knockdown/knockout controls: When available, include MRPS28 knockdown/knockout samples to validate antibody specificity

• Sample preparation optimization:

  • For mitochondrial proteins like MRPS28, consider using mitochondrial enrichment protocols

  • Optimize protein extraction methods based on subcellular localization (mitochondrial)

  • For ICC/IF, fixation and permeabilization conditions may significantly impact epitope recognition

• Antibody validation strategy:

  • Cross-validate results using multiple antibodies targeting different epitopes of MRPS28

  • Consider orthogonal methods to confirm antibody-based findings (e.g., mass spectrometry, RNA expression)

• Variables and relationship analysis:

  • Clearly define independent variables (e.g., treatment conditions) and dependent variables (e.g., MRPS28 expression levels)

  • Control for confounding variables that might affect mitochondrial function or protein expression

Implementing these considerations will strengthen experimental design and increase the reliability of results when working with MRPS28 antibodies.

What approaches can be used to validate MRPS28 antibody specificity?

Validating antibody specificity is crucial for generating reliable research data. For MRPS28 antibodies, consider the following validation approaches:

• Molecular weight verification:

  • MRPS28 has a calculated molecular weight of approximately 20.8 kDa

  • Verify that the detected band in Western blots corresponds to this expected size

• Genetic approaches:

  • siRNA/shRNA knockdown of MRPS28 to confirm reduced signal

  • CRISPR/Cas9-mediated knockout as a definitive negative control

  • Overexpression systems to confirm increased antibody signal

• Peptide competition assays:

  • Pre-incubate the antibody with the immunizing peptide/protein

  • This should block specific binding and eliminate true positive signals

• Cross-species reactivity testing:

  • Test the antibody in multiple species if it claims cross-reactivity (e.g., human and mouse)

  • Compare observed patterns with predicted evolutionary conservation

• Orthogonal detection methods:

  • Correlate antibody results with mRNA expression data

  • Compare with mass spectrometry proteomics data where available

  • Use multiple antibodies targeting different epitopes of MRPS28

• Application-specific validation:

  • For each application (WB, ICC/IF), perform specific validation tests

  • Include appropriate positive and negative controls for each experiment type

Thorough validation ensures that experimental findings truly reflect MRPS28 biology rather than non-specific antibody interactions.

How can researchers troubleshoot non-specific binding or weak signals with MRPS28 antibodies?

When encountering issues with MRPS28 antibodies, systematic troubleshooting is essential:

• Addressing non-specific binding:

  • Increase blocking stringency: Use 5% BSA or milk in TBS-T for Western blots

  • Optimize antibody dilution: Test a range around the recommended dilution (e.g., 1:250 to 1:2000 for WB)

  • Add detergents: Include 0.1-0.3% Triton X-100 in washing buffers

  • Perform additional washes: Increase wash duration and number of wash steps

  • Use highly purified antibodies: Consider protein A-purified antibodies when available

• Resolving weak signal issues:

  • Increase protein loading: For Western blots, load more total protein

  • Decrease antibody dilution: Use a more concentrated antibody solution

  • Optimize incubation conditions: Extend primary antibody incubation to overnight at 4°C

  • Enhance detection systems: Use signal amplification methods like TSA (tyramide signal amplification)

  • Modify sample preparation: Use fresh samples and optimize extraction buffers for mitochondrial proteins

• Application-specific troubleshooting:

  • For Western blots: Adjust transfer conditions, membrane type, and blocking reagents

  • For ICC/IF: Optimize fixation methods, antigen retrieval, and permeabilization conditions

• Data analysis approach:

  • Compare results between samples processed in the same experiment

  • Document all experimental conditions systematically

  • Consider statistical analysis to distinguish signal from noise

Systematic troubleshooting can identify optimal conditions for specific experimental systems, resulting in more reliable and reproducible data.

What are the advanced applications of MRPS28 antibodies in mitochondrial research?

MRPS28 antibodies can be employed in several sophisticated applications beyond standard detection methods:

• Co-immunoprecipitation (Co-IP) studies:

  • Investigate protein-protein interactions involving MRPS28

  • Identify novel binding partners within the mitochondrial ribosome

  • Study dynamics of mitochondrial ribosome assembly

• Chromatin immunoprecipitation (ChIP):

  • Although primarily a mitochondrial protein, potential nuclear interactions can be studied

  • Investigate whether MRPS28 has any role in nuclear-mitochondrial communication

• Proximity labeling approaches:

  • Combine with BioID or APEX2 to identify proteins in close proximity to MRPS28

  • Map the local proteome within the mitochondrial ribosome

• Super-resolution microscopy:

  • Study the precise localization of MRPS28 within mitochondrial structures

  • Investigate co-localization with other mitochondrial ribosomal proteins

• Live-cell imaging:

  • When combined with genetically encoded tags, antibodies can validate expression patterns

  • Study dynamics of mitochondrial ribosome assembly in living cells

• Tissue microarray analysis:

  • Examine MRPS28 expression across multiple tissue types

  • Investigate correlation with disease states or developmental stages

• Single-cell analysis:

  • Combine with single-cell technologies to study heterogeneity in MRPS28 expression

  • Correlate with mitochondrial function at the single-cell level

These advanced applications enable researchers to gain deeper insights into MRPS28 function and its role in mitochondrial translation and cellular metabolism.

How can researchers interpret contradictory results when using different MRPS28 antibodies?

When different MRPS28 antibodies yield contradictory results, a systematic analysis approach is necessary:

• Epitope mapping and comparison:

  • Determine the exact epitopes recognized by each antibody

  • Consider whether different antibodies target distinct domains of MRPS28

  • Evaluate potential post-translational modifications that might affect epitope recognition

• Critical evaluation of antibody validation:

  • Review validation data for each antibody

  • Assess the rigor of specificity testing conducted by manufacturers

  • Consider independent validation using genetic approaches (knockdown/knockout)

• Experimental design analysis:

  • Document all experimental variables: buffers, incubation times, detection methods

  • Control for batch effects by running comparative experiments concurrently

  • Consider blind analysis of results to prevent experimenter bias

• Sample-specific considerations:

  • Evaluate whether contradictions are tissue/cell-type specific

  • Consider species differences if working across multiple organisms

  • Assess potential sample preparation artifacts

• Resolution strategies:

  Strategy	Implementation
  Orthogonal validation	Use non-antibody methods (mass spectrometry, RNA analysis)
  Multiple antibody approach	Use at least three antibodies targeting different epitopes
  Genetic validation	Perform experiments in knockout/knockdown systems
  Independent replication	Have different researchers/labs replicate key experiments

• Publication and reporting:

  • Transparently report contradictory results in publications

  • Provide detailed methods including catalog numbers and lot numbers

  • Consider pre-registering key experiments to reduce publication bias

By systematically analyzing contradictory results, researchers can gain deeper insights into MRPS28 biology and potentially discover novel aspects of protein function, isoforms, or modifications.

What are the optimal sample preparation protocols for detecting MRPS28 in different applications?

Sample preparation significantly impacts MRPS28 detection across different applications:

• Western Blot sample preparation:

  • Whole cell lysates: Use RIPA buffer supplemented with protease inhibitors

  • Mitochondrial enrichment: Consider using mitochondrial isolation kits for enhanced detection

  • Protein denaturation: Heat samples to 95°C for 5 minutes in reducing sample buffer

  • Loading amount: Start with 20-30 μg total protein (adjust based on expression level)

• Immunocytochemistry/Immunofluorescence preparation:

  • Fixation: 4% paraformaldehyde (10-15 minutes) preserves structure while maintaining epitope accessibility

  • Permeabilization: 0.1-0.3% Triton X-100 (10 minutes) allows antibody access to mitochondrial targets

  • Blocking: 5% normal serum (from secondary antibody host species) for 1 hour

  • Mitochondrial co-staining: Consider co-staining with MitoTracker or anti-TOMM20 to confirm mitochondrial localization

• Co-immunoprecipitation preparation:

  • Use gentler lysis buffers (e.g., NP-40 buffer) to preserve protein-protein interactions

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Consider crosslinking for transient interactions

  • Include RNase treatment controls to distinguish RNA-mediated from direct protein interactions

• Tissue section preparation:

  • Fixation: 10% neutral buffered formalin followed by paraffin embedding

  • Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Section thickness: 4-6 μm sections for optimal antibody penetration

  • Blocking: Include both protein blocking and endogenous peroxidase blocking steps

Optimization of these protocols for specific experimental systems will maximize detection sensitivity and specificity for MRPS28.

What are the recommended quantification methods for MRPS28 expression analysis?

Accurate quantification of MRPS28 expression requires appropriate methods for different experimental approaches:

• Western blot quantification:

  • Densitometry software: Use ImageJ, Image Lab, or similar software for band intensity analysis

  • Normalization strategies:

    • Normalize to loading controls (β-actin, GAPDH)

    • For mitochondrial studies, normalize to mitochondrial markers (VDAC, COX IV)

  • Standard curve approach: Include a dilution series of positive control for absolute quantification

  • Linear dynamic range: Ensure signal is within the linear range of detection

• Immunofluorescence quantification:

  • Mean fluorescence intensity: Measure average pixel intensity within defined cellular regions

  • Integrated density: Calculate the product of area and mean intensity

  • Co-localization analysis: Use Pearson's or Mander's coefficient to quantify overlap with mitochondrial markers

  • Single-cell analysis: Quantify cell-to-cell variation in expression levels

• qPCR for mRNA expression:

  • Use as a complementary approach to protein analysis

  • Select appropriate reference genes (ideally multiple)

  • Apply the ΔΔCt method for relative quantification

  • Consider absolute quantification with standard curves for more precise measurements

• Systematic error reduction:

  • Include technical replicates (minimum 3)

  • Process all compared samples in the same experiment

  • Use blinded analysis to reduce experimenter bias

  • Apply appropriate statistical tests for the experimental design

Combining multiple quantification approaches provides the most comprehensive and reliable assessment of MRPS28 expression levels.

How should researchers design experiments to study MRPS28 interactions with other mitochondrial proteins?

Studying protein-protein interactions involving MRPS28 requires careful experimental design:

• Co-immunoprecipitation (Co-IP) approach:

  • Use antibodies against MRPS28 to pull down protein complexes

  • Perform reciprocal Co-IPs with antibodies against suspected interaction partners

  • Include appropriate negative controls (IgG control, irrelevant antibody)

  • Consider native vs. denaturing conditions based on interaction stability

• Proximity labeling methods:

  • Generate MRPS28 fusion proteins with BioID or APEX2

  • Identify nearby proteins through biotinylation and streptavidin pulldown

  • Compare results across different cellular conditions

  • Validate key interactions with orthogonal methods

• Fluorescence-based interaction studies:

  • FRET (Förster Resonance Energy Transfer): Detect direct protein interactions

  • BiFC (Bimolecular Fluorescence Complementation): Visualize interactions in living cells

  • PLA (Proximity Ligation Assay): Detect proteins within 40 nm distance using antibodies

• Genetic perturbation strategies:

  • Knockdown/knockout MRPS28 and assess effects on interacting proteins

  • Use domain mapping to identify specific interaction regions

  • Create targeted mutations to disrupt specific interactions

  • Employ rescue experiments with wildtype vs. mutant MRPS28

• Structural biology approaches:

  • Complement interaction studies with available structural data

  • Consider cryo-EM studies of mitochondrial ribosomes

  • Use computational modeling to predict interaction interfaces

• Controls and validation:

  • Include known mitochondrial ribosomal protein interactions as positive controls

  • Use non-interacting mitochondrial proteins as negative controls

  • Validate novel interactions with multiple independent methods

  • Consider RNA-dependence of interactions within ribosomal complexes

A multi-method approach with appropriate controls provides the strongest evidence for genuine MRPS28 protein interactions.

What are the key considerations for using MRPS28 antibodies in disease-related research?

When investigating MRPS28 in disease contexts, researchers should consider:

• Disease-specific expression patterns:

  • Compare MRPS28 expression between normal and disease tissues

  • Correlate expression levels with disease progression or patient outcomes

  • Consider potential post-translational modifications in disease states

  • Analyze subcellular localization changes in pathological conditions

• Experimental design for disease models:

  • Select appropriate disease models (cell lines, animal models, patient samples)

  • Include sufficient biological replicates to account for heterogeneity

  • Match cases and controls for confounding variables (age, sex, treatment)

  • Consider longitudinal studies to track changes over disease progression

• Technical considerations:

  • Optimize fixation and antigen retrieval for clinical samples

  • Validate antibody performance specifically in disease-relevant tissues

  • Consider automated staining systems for consistency across samples

  • Use quantitative image analysis for objective assessment

• Clinicopathological correlations:

  • Correlate MRPS28 alterations with clinical parameters

  • Consider multivariate analysis to identify independent associations

  • Evaluate potential as diagnostic or prognostic marker

  • Assess relationship to treatment response or resistance

• Mechanistic investigations:

  • Determine whether MRPS28 alterations are causal or consequential

  • Investigate effects on mitochondrial translation

  • Assess impact on cellular metabolism and energy production

  • Evaluate potential as therapeutic target

• Ethical and practical considerations:

  • Ensure appropriate ethical approvals for human samples

  • Consider sample size requirements for statistical power

  • Plan for long-term storage of valuable samples

  • Document detailed clinical data associated with samples

Careful attention to these considerations will enhance the translational relevance of MRPS28 research in disease contexts.

What are the technical specifications of commonly used MRPS28 antibodies?

The following table summarizes key technical specifications of representative MRPS28 antibodies:

This information should guide selection of the appropriate antibody based on specific experimental requirements and target species .

What bioinformatic resources are available for MRPS28 research?

Researchers can leverage various bioinformatic resources to enhance MRPS28 studies:

• Sequence and structural databases:

  • UniProt: Comprehensive protein information (ID: P82673 for human MRPS28)

  • PDB: Structural data for mitochondrial ribosomes including MRPS28

  • Ensembl: Genomic and transcript information (ENSG00000147586)

  • NCBI Gene: Comprehensive gene information (ID: 28957)

• Expression databases:

  • Human Protein Atlas: Tissue expression patterns of MRPS28

  • GTEx Portal: Tissue-specific expression data

  • GEO: Gene expression datasets for various conditions

  • Single Cell Expression Atlas: Cell-type specific expression

• Interaction databases:

  • STRING: Protein-protein interaction network

  • BioGRID: Curated interaction data

  • IntAct: Molecular interaction database

  • Pharos: Target knowledge database (shows high knowledge value for interacting proteins: 0.98)

• Disease associations:

  • DisGeNET: Gene-disease associations

  • OMIM: Mendelian disorders related to mitochondrial function

  • TCGA: Cancer genomics data

• Functional annotation resources:

  • Gene Ontology: Functional classifications

  • KEGG: Pathway information

  • Reactome: Detailed pathway data

• Prediction tools:

  • MitoProt: Mitochondrial targeting prediction

  • TargetP: Subcellular localization prediction

  • MitoMiner: Integrated mitochondrial protein database

These resources provide valuable context for experimental studies and can help generate new hypotheses regarding MRPS28 function and regulation.

How can researchers evaluate and compare different MRPS28 antibodies based on validation data?

Systematic evaluation of antibody validation data is essential for selecting the most appropriate MRPS28 antibody:

• Validation data assessment matrix:

• Critical evaluation of validation images:

  • Examine full blot images (not cropped) to assess background and specificity

  • Check for appropriate positive and negative controls

  • Evaluate signal-to-noise ratio in immunostaining images

  • Assess subcellular localization pattern (should be mitochondrial for MRPS28)

• Publication track record:

  • Search for antibody catalog numbers in literature

  • Review methods and results from published studies

  • Contact authors of key papers for feedback on antibody performance

  • Consider reproducibility across different research groups

• Independent validation approaches:

  • Test antibody performance in house before full studies

  • Validate with orthogonal methods (mass spectrometry, RNA expression)

  • Consider antibody validation initiatives (e.g., Antibodypedia)

  • Evaluate antibody ranking in community resources

• Decision framework:

  • Prioritize antibodies with multiple validation methods

  • Consider application-specific validation for your intended use

  • Evaluate cost-benefit for research-critical applications

  • Factor in reproducibility needs for long-term projects

Thorough evaluation of validation data increases confidence in antibody performance and experimental results reliability.

How is MRPS28 research contributing to our understanding of mitochondrial diseases?

MRPS28 research is providing new insights into mitochondrial disease mechanisms:

• Mitochondrial translation defects:

  • As a component of the mitochondrial ribosome, MRPS28 dysfunction may impact translation of mitochondrially-encoded proteins

  • This can affect oxidative phosphorylation and energy production

  • Research using MRPS28 antibodies helps characterize ribosome assembly defects in patient samples

• Disease associations:

  • Emerging evidence links MRPS28 to mitochondrial disease phenotypes

  • Variations in MRPS28 may contribute to combined oxidative phosphorylation deficiency (COXPD)

  • Studies are investigating potential connections to neurodegenerative conditions with mitochondrial involvement

• Tissue-specific effects:

  • Differential expression of MRPS28 across tissues may explain tissue-specific disease manifestations

  • High-energy demanding tissues (brain, heart, muscle) may be particularly sensitive to MRPS28 dysfunction

  • Immunohistochemistry with MRPS28 antibodies enables tissue-specific expression profiling

• Therapeutic implications:

  • Understanding MRPS28 function may reveal new therapeutic targets

  • Small molecules targeting mitochondrial translation show promise in preclinical studies

  • Antibody-based detection methods are essential for evaluating treatment effects on mitochondrial ribosome integrity

• Diagnostic applications:

  • MRPS28 antibodies may serve as diagnostic tools for certain mitochondrial disorders

  • Expression patterns might correlate with disease severity or progression

  • Combined with other markers, MRPS28 status could improve diagnostic accuracy

Ongoing research using MRPS28 antibodies continues to expand our understanding of mitochondrial disease mechanisms and potential therapeutic approaches.

What are the latest methodological advances in MRPS28 antibody-based research?

Recent technological and methodological advances are enhancing MRPS28 antibody applications:

• Multiplexed imaging approaches:

  • Cyclic immunofluorescence (CycIF) allows detection of >40 proteins in the same sample

  • Mass cytometry imaging (IMC) enables antibody-based detection with metal isotope labels

  • These approaches permit simultaneous analysis of MRPS28 with multiple other proteins

• Super-resolution microscopy advances:

  • STORM, PALM, and STED microscopy provide nanoscale resolution of mitochondrial structures

  • Expansion microscopy physically enlarges specimens for enhanced resolution

  • These techniques reveal precise MRPS28 localization within mitochondrial compartments

• Single-cell proteomics integration:

  • Combination of antibody-based detection with single-cell RNA-seq

  • CITE-seq and REAP-seq allow simultaneous protein and RNA profiling

  • These approaches correlate MRPS28 protein levels with transcriptome-wide effects

• Spatial transcriptomics correlation:

  • Integration of antibody staining with spatial transcriptomics

  • Correlates MRPS28 protein distribution with local transcriptome profiles

  • Provides insights into regional variation in mitochondrial function

• Automated high-throughput screening:

  • Automated immunostaining and imaging platforms

  • Machine learning-based image analysis of MRPS28 patterns

  • Enables large-scale screening of genetic or chemical perturbations

• Antibody engineering improvements:

  • Recombinant antibody technologies with improved reproducibility

  • Nanobodies and single-domain antibodies for enhanced penetration

  • Site-specific conjugation methods for better imaging probe attachment

These methodological advances are expanding the capabilities of MRPS28 antibody-based research, enabling more detailed and comprehensive studies of mitochondrial function.