MRPL28 Antibody

What is MRPL28 and why is it important in research?

MRPL28 is a mitochondrial ribosomal protein that functions as part of the large 39S subunit of mitoribosomes. It plays a critical role in mitochondrial protein synthesis. Mitochondrial ribosomes are distinct from cytoplasmic ribosomes, containing approximately 75% protein to rRNA (compared to prokaryotic ribosomes where this ratio is reversed). MRPL28 was originally isolated based on its ability to recognize tyrosinase in an HLA-A24-restricted fashion, suggesting potential roles in immune recognition . The protein is also known by alternative names including L28mt, MAAT1, Melanoma antigen p15, and MRP-L28 .

What are the validated applications for MRPL28 antibodies?

MRPL28 antibodies have been validated for multiple applications with specific recommended dilutions:

These applications have been successfully tested across multiple cell lines including HEK-293T, HeLa, HepG2, A375, and JAR cells .

What is the molecular weight of MRPL28 and how does this affect antibody selection?

The calculated molecular weight of MRPL28 is 30 kDa, which has been consistently observed in Western blot analyses . When selecting an antibody, it's important to verify this expected band size in validation data and consider that post-translational modifications might affect migration patterns. Always run appropriate positive controls (such as HeLa or HepG2 cell lysates) alongside experimental samples to confirm specificity .

How should I determine the optimal antibody dilution for my specific experimental system?

The optimal antibody dilution varies based on application and specific sample type. To determine the ideal concentration:

• Start with a dilution titration experiment using the manufacturer's recommended range (e.g., 1:1000-1:4000 for WB)

• Use positive control samples with known MRPL28 expression (HEK-293T, HeLa, HepG2 cells are well-validated)

• For Western blots, include protein loading controls

• For immunostaining, include secondary antibody-only controls to assess background

• Consider cell/tissue-specific factors that might affect antigen accessibility

As noted in multiple protocols, "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" .

What are the recommended antigen retrieval methods for MRPL28 IHC?

For optimal IHC results with MRPL28 antibodies, antigen retrieval methods have been validated:

• Primary recommendation: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

The choice between these methods should be based on empirical testing with your specific tissue samples. Paraffin-embedded tissues, particularly human kidney and testis tissues, have been successfully stained using these protocols .

How can I ensure specificity when using MRPL28 antibodies for co-localization studies?

For reliable co-localization studies:

• Use a fluorescently conjugated MRPL28 antibody (such as CoraLite® Plus 488) at the validated dilution of 1:50-1:500

• Pair with mitochondrial markers (e.g., TOMM20, COX IV) to confirm mitochondrial localization

• Include single-staining controls to assess bleed-through

• Validate specificity by comparing staining patterns in positive control cells (HeLa cells have been well-characterized)

• Consider z-stack acquisition to accurately assess three-dimensional co-localization

Multiple studies have successfully demonstrated mitochondrial localization patterns using validated MRPL28 antibodies in immunofluorescence applications .

How can I use MRPL28 antibodies to investigate mitochondrial ribosome assembly in disease models?

To study mitochondrial ribosome assembly:

• Co-immunoprecipitation approach: Use IP-validated MRPL28 antibodies (0.5-4.0 μg for 1.0-3.0 mg of protein lysate) to pull down associated mitoribosome components

• Sucrose gradient fractionation: Separate mitoribosomal complexes and detect MRPL28 with validated antibodies (1:1000-1:4000 dilution for WB)

• Proximity labeling: Combine with BioID or APEX2 approaches to identify proximal proteins

• Cross-comparison: Analyze samples from control and disease states to identify assembly defects

Mouse brain tissue has been successfully used for IP applications with MRPL28 antibodies , making it suitable for comparative studies between healthy and disease models.

What controls should I include when investigating tissue-specific expression patterns of MRPL28?

For rigorous tissue expression analysis:

• Positive tissue controls: Include kidney and testis tissues which consistently show positive staining

• Cell line controls: Include HeLa or HepG2 cells as positive controls

• Antibody validation controls:

  • Primary antibody omission

  • Isotype control antibody

  • Peptide competition assay where available

• Cross-validation: Compare results using antibodies from different vendors or targeting different epitopes

• Technical replicates: Process multiple sections to account for staining variability

When comparing across tissues, consistent antigen retrieval methods must be maintained for valid comparisons .

How can contradictory MRPL28 antibody staining patterns be reconciled in research?

When faced with conflicting staining patterns:

• Evaluate antibody characteristics: Compare epitopes, clonality (monoclonal vs. polyclonal), and host species

• Cross-validate with orthogonal methods: Confirm protein expression with RNA-seq data or multiple antibodies

• Check fixation and processing effects: Different fixatives can affect epitope accessibility

• Consider post-translational modifications: These may mask epitopes in certain contexts

• Evaluate tissue/cell heterogeneity: Expression may vary within tissues or cell populations

For example, comparing monoclonal (ab126719) versus polyclonal (ab196842) antibodies can provide complementary data, as monoclonals offer higher specificity while polyclonals may provide stronger signals through multiple epitope recognition .

What are the most common causes of non-specific background in MRPL28 immunostaining, and how can they be addressed?

Common causes of non-specific background include:

• Excessive antibody concentration: Titrate to determine optimal dilution (starting with 1:200-1:800 for IF/ICC)

• Insufficient blocking: Extend blocking time or increase blocking agent concentration (BSA or serum)

• Cross-reactivity: Validate in MRPL28-knockout samples or with peptide competition assays

• Insufficient washing: Increase washing steps duration and volume

• Autofluorescence (for IF): Include unstained controls and consider autofluorescence quenching methods

For paraffin-embedded tissues, background can be particularly challenging. Validated protocols recommend antigen retrieval with TE buffer pH 9.0 followed by peroxidase blocking for IHC applications .

How should storage conditions be modified for long-term preservation of MRPL28 antibody activity?

For optimal long-term storage:

• Store at -20°C in manufacturer-recommended buffer (typically PBS with 0.02% sodium azide and 50% glycerol, pH 7.3)

• For antibodies containing BSA (e.g., 0.1% BSA), avoid repeated freeze-thaw cycles which can cause protein aggregation

• If frequent use is needed, prepare working aliquots to minimize freeze-thaw cycles

• Protect fluorescently conjugated antibodies (like CoraLite® Plus 488) from light exposure

• Monitor storage buffer pH periodically, as pH shifts can affect antibody stability

While some manufacturers note that "Aliquoting is unnecessary for -20°C storage" , this applies to specific formulations and may not be universal for all MRPL28 antibodies.

How can I resolve discrepancies between expected and observed molecular weights in Western blot applications?

When facing molecular weight discrepancies:

• Confirm sample preparation: Ensure complete denaturation and reduction of samples

• Verify gel percentage: Use appropriate percentage for 30 kDa proteins (10-12% typically optimal)

• Check for post-translational modifications: Phosphorylation, glycosylation, or ubiquitination can alter migration

• Evaluate proteolytic processing: MRPL28 may undergo specific cleavage in certain contexts

• Cross-validate with multiple antibodies: Different epitopes may reveal different processed forms

Multiple validated antibodies consistently detect MRPL28 at its expected 30 kDa size in various human and mouse samples , providing a reliable reference point for troubleshooting.

How do rabbit monoclonal and polyclonal MRPL28 antibodies compare in sensitivity and specificity across applications?

Comparative analysis reveals:

Research has shown that recombinant antibodies appear to be on average higher in affinity compared to traditional monoclonal antibodies , offering advantages for certain applications.

What methodological considerations are important when using MRPL28 antibodies for analyzing mitochondrial stress responses?

For mitochondrial stress analyses:

• Experimental timing: Collect samples at multiple timepoints post-stress induction

• Subcellular fractionation: Compare whole-cell versus purified mitochondrial fractions for MRPL28 levels

• Co-staining approach: Pair MRPL28 with mitochondrial stress markers (e.g., HSP60, PINK1)

• Quantification methods: Use digital image analysis for precise quantification of signal intensity changes

• Controls: Include both positive stress controls (CCCP, rotenone) and negative controls

Validated antibodies with confirmed mitochondrial localization patterns should be selected, with CoraLite® Plus 488-conjugated antibodies offering advantages for live-cell imaging applications .

How can MRPL28 antibodies be integrated into multi-omics approaches for comprehensive mitochondrial function studies?

For multi-omics integration:

• Proteomics verification: Use IP with MRPL28 antibodies followed by mass spectrometry to identify interacting partners

• Transcriptomics correlation: Compare MRPL28 protein levels (by WB) with mRNA expression data

• Structural biology approaches: Combine with cryo-EM studies of mitoribosome complexes

• Metabolomics connections: Correlate MRPL28 expression/localization changes with metabolic shifts

• Systems biology modeling: Incorporate antibody-derived localization/interaction data into predictive models

Recent work on molecular surface descriptors for antibody developability can inform selection of optimal antibodies for integrated studies, particularly when examining interactions or developing new detection methodologies.

How can MRPL28 antibodies be utilized in investigating the crosstalk between mitochondrial translation and nuclear gene expression?

For studying mito-nuclear communication:

• Dual immunofluorescence: Use MRPL28 antibodies (1:200-1:800 dilution) alongside nuclear-encoded mitochondrial protein markers

• Subcellular fractionation: Compare MRPL28 levels in mitochondrial vs. non-mitochondrial fractions during cellular stress

• Proximity ligation assay (PLA): Detect potential interactions between MRPL28 and signaling molecules

• ChIP-seq correlation: Compare chromatin immunoprecipitation data with MRPL28 expression patterns

• Time-course analysis: Monitor dynamic changes during mitochondrial biogenesis or stress responses

This approach provides insights into how mitochondrial translation machinery components like MRPL28 might influence or respond to nuclear signaling pathways .

What considerations are important when using MRPL28 antibodies for studying mitochondrial heterogeneity in complex tissues?

For analyzing mitochondrial heterogeneity:

• Single-cell resolution techniques: Combine with laser capture microdissection or single-cell Western blot

• Multiplexed immunofluorescence: Pair MRPL28 with cell-type markers and other mitochondrial proteins

• Super-resolution microscopy: Employ techniques like STORM or STED for sub-mitochondrial localization

• Tissue-specific controls: Include region-matched control tissues (kidney and testis tissues are well-validated)

• Quantitative image analysis: Develop algorithms to assess MRPL28 staining intensity variations across tissue regions

These approaches can reveal tissue-specific differences in mitochondrial composition and function, particularly important in heterogeneous organs like brain and kidney .

How might dynamic changes in MRPL28 expression be monitored during cellular differentiation or disease progression?

For temporal monitoring of MRPL28:

• Developmental time-course: Sample tissues/cells at defined developmental stages

• Disease progression models: Collect samples at multiple disease stages

• Quantitative Western blot: Use validated MRPL28 antibodies (1:1000-1:4000 dilution) with appropriate normalization

• High-content imaging: Analyze population-level changes in MRPL28 immunofluorescence patterns

• Correlation with functional assays: Pair MRPL28 detection with mitochondrial activity measurements