MRRF Antibody

What is MRRF and what is its role in cellular function?

MRRF (Mitochondrial Ribosome Recycling Factor) is a protein component of the mitochondrial translational machinery. It functions primarily in the mitochondrial matrix and is essential for protein synthesis within mitochondria . MRRF has a calculated molecular weight of 29 kDa, though it is sometimes observed at 25 kDa in experimental systems . The protein is encoded by the MRRF gene (also known as RRF, MRFF, or MTRRF) with Gene ID 92399 and UniProt ID Q96E11 . Functionally, MRRF is involved in the recycling of mitochondrial ribosomes after the completion of protein synthesis, a critical step for maintaining mitochondrial translation efficiency.

What applications are MRRF antibodies validated for?

MRRF antibodies have been validated for several research applications as shown in the following table:

These applications allow researchers to detect and quantify MRRF protein expression in various experimental contexts, with Western blot being the most thoroughly validated application across different antibodies.

What sample types show positive results with MRRF antibodies?

Various cell lines and tissue samples have been validated for MRRF antibody detection:

This diverse range of validated samples indicates that MRRF is expressed across multiple cell and tissue types, making these antibodies versatile tools for both human and rodent studies.

What are the specific technical details of commercially available MRRF antibodies?

Researchers should be aware of the following technical specifications when selecting an MRRF antibody:

Understanding these technical details is critical for experimental design and interpretation, particularly when troubleshooting or comparing results across different studies .

How should MRRF antibodies be stored to maintain reactivity?

Proper storage is essential for maintaining antibody performance. For the 12357-2-AP MRRF antibody, the manufacturer recommends storage at -20°C, where it remains stable for one year after shipment . The antibody is provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, and aliquoting is unnecessary for -20°C storage . Some preparations (20μL sizes) contain 0.1% BSA, which helps stabilize dilute antibody solutions . While specific storage information for CAB2443 is not provided in the search results, similar storage conditions are likely appropriate. Researchers should avoid repeated freeze-thaw cycles, which can degrade antibody quality and affect experimental reproducibility.

How should researchers validate MRRF antibody specificity?

Antibody validation is critical for ensuring experimental reproducibility, particularly in light of what has been termed the "antibody characterization crisis" . For MRRF antibodies, consider implementing these validation strategies:

• Genetic controls: Use MRRF knockout or knockdown samples to confirm signal specificity.

• Multiple antibody approach: Validate key findings with different antibodies targeting distinct MRRF epitopes.

• Peptide competition: Pre-incubate the antibody with excess immunizing peptide to demonstrate specific binding.

• Cross-reactivity testing: Test antibody performance in samples known to express or lack MRRF.

• Band size verification: Confirm detection at the expected molecular weight (calculated 29 kDa, observed 25-29 kDa) .

The broader scientific community has recognized that "many antibodies have not been adequately characterized, which casts doubt on the results reported in many scientific papers" . Therefore, implementing rigorous validation protocols is essential for generating reliable data with MRRF antibodies.

How does MRRF expression correlate with mitochondrial function?

While the search results don't provide direct experimental data on this correlation, they reference publications linking MRRF to mitochondrial biogenesis and function . One referenced study examines how "muscle denervation reduces mitochondrial biogenesis and mitochondrial translation factor expression in mice," while another investigates "exercise-induced mitochondrial biogenesis" . These references suggest that MRRF expression may be regulated in coordination with other mitochondrial translation factors during conditions that alter mitochondrial function or biogenesis.

For researchers interested in this correlation, experimental approaches might include:

How do different antibody formats impact MRRF detection methods?

The search results describe both polyclonal (12357-2-AP) and monoclonal (CAB2443) antibodies against MRRF . These different formats have distinct advantages and limitations:

For critical experiments, validation with both antibody types may provide complementary information and increase confidence in results.

What methodological considerations are important for studying MRRF in disease models?

When investigating MRRF in disease contexts, several methodological considerations become critical:

• Appropriate controls: Include age-matched, sex-matched controls for disease models, as mitochondrial function can vary with these parameters.

• Tissue specificity: MRRF expression and function may differ between tissues, as evidenced by the different validation samples listed . This tissue-specific expression should be considered when designing experiments.

• Subcellular fractionation quality: Since MRRF is localized to mitochondria , the quality of mitochondrial isolation can significantly impact detection sensitivity and specificity.

• Quantification methods: When comparing MRRF levels between normal and disease states, standardized quantification methods with appropriate normalization controls are essential.

• Functional correlates: Pair MRRF expression analysis with functional assays of mitochondrial translation to establish mechanistic links between altered MRRF levels and disease phenotypes.

What are the optimal protocols for Western blot detection of MRRF?

Based on the search results, the following protocol elements are recommended for Western blot detection of MRRF:

For optimal results, researchers should:

• Titrate antibody concentrations: The wide dilution range suggested (1:2000-1:10000 for 12357-2-AP) indicates that optimization for specific experimental conditions is necessary .

• Include positive controls: Using validated cell lines such as HEK-293 cells helps confirm antibody performance .

• Verify band size: The observed molecular weight may vary slightly (25-29 kDa) depending on experimental conditions and sample types .

• Follow manufacturer protocols: Specific Western blot protocols are available from manufacturers for optimal results .

What are the recommended protocols for immunohistochemistry using MRRF antibodies?

For immunohistochemistry applications with MRRF antibodies, the following protocol elements are recommended:

Successful IHC detection of MRRF requires careful attention to:

• Appropriate antigen retrieval: TE buffer at pH 9.0 is recommended, with citrate buffer at pH 6.0 as an alternative .

• Antibody titration: The wide dilution range (1:20-1:200) suggests that optimization is necessary for specific tissue types and fixation methods .

• Controls: Include positive control tissues (e.g., human pancreas cancer tissue) to validate the staining procedure .

• Detection systems: While not specified in the search results, compatible secondary detection systems should be selected based on the host species (rabbit) and experimental requirements.

How can researchers troubleshoot non-specific binding with MRRF antibodies?

Non-specific binding can compromise experimental results. When troubleshooting MRRF antibody experiments:

• Optimize blocking: Use appropriate blocking agents (BSA, non-fat milk) and ensure sufficient blocking time.

• Adjust antibody concentration: The wide recommended dilution ranges (e.g., 1:2000-1:10000 for Western blot with 12357-2-AP) suggest that titration is essential for finding the optimal signal-to-noise ratio .

• Increase washing stringency: More thorough washing steps can help reduce non-specific binding.

• Validate with genetic controls: Use MRRF-deficient samples as negative controls to identify non-specific bands.

• Secondary antibody controls: Include controls omitting primary antibody to identify non-specific secondary antibody binding.

• Consider tissue-specific optimization: Different tissues may require different optimization strategies, as suggested by the diverse validation samples listed .

What quantification methods are appropriate for MRRF expression analysis?

For accurate quantification of MRRF expression:

• Appropriate loading controls: For whole-cell lysates, housekeeping proteins like β-actin or GAPDH are suitable. For mitochondrial fractions, mitochondrial markers such as VDAC or COX IV provide better normalization.

• Densitometry software: Use standardized analysis software with background subtraction capabilities for Western blot quantification.

• Linear dynamic range: Ensure signal intensity falls within the linear dynamic range of detection for accurate quantification.

• Technical replicates: Perform multiple technical replicates to account for variability in blotting and detection.

• Standard curves: For absolute quantification, consider including recombinant MRRF standards at known concentrations.

• Statistical analysis: Apply appropriate statistical tests based on experimental design and data distribution.

How should researchers interpret changes in MRRF expression in experimental models?

Interpreting changes in MRRF expression requires consideration of several factors:

• Context of mitochondrial function: MRRF changes should be interpreted alongside other markers of mitochondrial function and biogenesis .

• Magnitude of change: Establish what constitutes biologically significant changes in MRRF expression through controlled experiments.

• Tissue-specific baseline expression: Consider that baseline MRRF expression may vary between tissues, as evidenced by the different validation samples .

• Protein vs. mRNA levels: Compare protein expression changes with mRNA levels to distinguish between transcriptional and post-transcriptional regulation.

• Functional consequences: Correlate MRRF expression changes with functional outcomes related to mitochondrial translation to establish physiological significance.

• Disease relevance: In disease models, compare findings with human patient samples when possible to establish clinical relevance.

How can researchers ensure reproducible results with MRRF antibodies?

Ensuring reproducibility with antibody-based research is particularly important given the "antibody characterization crisis" highlighted in recent literature . For MRRF antibody experiments:

• Use RRIDs: Research Resource Identifiers (e.g., AB_2144259 for 12357-2-AP) help track specific antibodies across studies and enhance reproducibility.

• Document all experimental details: Record antibody catalog numbers, lot numbers, dilutions, incubation times, and detection methods.

• Validate with multiple antibodies: Confirm key findings using different antibodies targeting different MRRF epitopes.

• Include appropriate controls: Use positive controls (e.g., validated cell lines) and negative controls (e.g., MRRF-depleted samples).

• Share detailed protocols: When publishing, provide comprehensive methodological details beyond standard descriptions.

• Pre-register critical experiments: Consider pre-registering key experimental protocols to enhance transparency.

How should researchers interpret conflicting results between different MRRF antibodies?

When faced with conflicting results between different MRRF antibodies:

• Review epitope information: Different antibodies target different regions of MRRF, which may be differentially affected by experimental conditions or protein interactions .

• Compare antibody formats: Results may differ between polyclonal and monoclonal antibodies due to their inherent characteristics.

• Assess validation evidence: Review the validation data for each antibody, including specificity controls and recommended applications.

• Consider post-translational modifications: Modifications within antibody epitopes may affect recognition and lead to apparently conflicting results.

• Evaluate experimental conditions: Different sample preparation methods, buffers, or detection systems may impact antibody performance.

• Use orthogonal methods: Employ non-antibody-based methods (e.g., mass spectrometry) to resolve discrepancies.

What are the common pitfalls in analyzing MRRF expression data?

Researchers should be aware of these common pitfalls when analyzing MRRF expression:

How does the "antibody characterization crisis" affect research with MRRF antibodies?

The "antibody characterization crisis" described in recent literature has significant implications for MRRF research :

• Questionable reproducibility: Many published findings using inadequately characterized antibodies may be difficult to reproduce.

• Validation burden: Researchers must invest substantial resources in validating commercial antibodies before use.

• Publication requirements: Journals increasingly require more rigorous antibody validation evidence.

• Resource repositories: The use of Research Resource Identifiers (RRIDs) like AB_2144259 helps track antibody use across studies.

• Methodology development: New validation approaches are being developed to address reproducibility challenges.

As noted in the literature, this crisis "casts doubt on the results reported in many scientific papers" and is "compounded by a lack of suitable control experiments in many studies" . Researchers working with MRRF antibodies should implement comprehensive validation strategies to ensure their findings are robust and reproducible.

What future developments might improve MRRF antibody research?

Several developments could enhance MRRF antibody research in the future:

• Improved validation standards: Implementation of more stringent industry and journal standards for antibody validation.

• Expanded epitope mapping: More detailed characterization of epitopes recognized by different MRRF antibodies.

• Genetic validation resources: Development of more accessible MRRF knockout/knockdown controls for validation.

• Alternative detection technologies: Non-antibody-based methods for MRRF detection and quantification.

• Structural insights: Better understanding of MRRF structure could inform epitope accessibility in different experimental conditions.

• Collaborative validation initiatives: Community-based efforts to systematically validate and benchmark MRRF antibodies, similar to ongoing efforts for other protein targets .

These developments would address the broader concerns about antibody reproducibility highlighted in recent literature while specifically enhancing the quality of MRRF-focused research.