MRFAP1 Antibody

What is MRFAP1 protein and why is it significant in cellular research?

MRFAP1 (Mof4 family associated protein 1) is a 14-15 kDa nuclear protein that plays critical roles in chromatin modification, cell cycle regulation, and mitotic progression. It functions by negatively regulating the recruitment of the NuA4 (nucleosome acetyltransferase of H4) histone acetyltransferase complex to chromatin . This protein is particularly significant due to its:

• Rapid turnover rate and regulation via the ubiquitin-proteasome system

• Cell cycle-dependent expression pattern (accumulated in metaphase, disappearing in anaphase)

• Complex formation with MORF4L1 and RB1

• Interaction with the Cul7/FBXW8 E3 ligase system

• Dramatic stabilization upon NEDD8 inhibition

• Tissue-specific expression patterns, notably in testis and brain

Research has demonstrated that appropriate regulation of MRFAP1 is essential for genomic stability, with overexpression causing growth retardation and severe mitotic cell death .

What are the optimal applications for MRFAP1 antibodies in research settings?

MRFAP1 antibodies have been validated for several research applications with specific optimization parameters:

The experimental design should incorporate appropriate positive controls and titration of antibody concentration for each specific application to achieve optimal results .

How should researchers design experiments to study MRFAP1's cell cycle-dependent regulation?

Designing experiments to study MRFAP1's cell cycle-dependent regulation requires careful synchronization protocols and multi-method validation:

• Cell Synchronization Approach:

  • Implement double thymidine block protocols to synchronize cells at G1/S boundary

  • Use nocodazole treatment (after thymidine release) to activate spindle checkpoint and prevent mitotic exit

  • Apply nocodazole block-and-release to specifically study anaphase-telophase transition

• Analytical Methods:

  • Combine flow cytometry (FACS) for DNA content measurement with Western blot analysis of MRFAP1 and cell cycle markers (such as Cyclin B1)

  • Apply immunofluorescence microscopy to visualize MRFAP1 localization across different mitotic stages (interphase, prophase, metaphase, anaphase, telophase)

  • Implement siRNA knockdown of FBXW8 to investigate the effect on MRFAP1 degradation during mitosis

• Controls and Validation:

  • Include cell cycle phase markers to verify synchronization efficiency

  • Monitor both endogenous and tagged MRFAP1 (using Flag-MRFAP1 stable cell lines)

  • Compare protein levels with mRNA expression to distinguish transcriptional from post-translational regulation

Research by Hu et al. demonstrated that MRFAP1 accumulates significantly in metaphase but completely disappears during anaphase, with reappearance in telophase—a pattern strictly regulated by FBXW8-mediated degradation .

What are the methodological considerations for studying MRFAP1's interaction with E3 ubiquitin ligases?

Investigating MRFAP1's interactions with E3 ubiquitin ligases requires specialized methodological approaches:

• Co-immunoprecipitation (Co-IP) Strategy:

  • Use MRFAP1 antibodies for pull-down experiments followed by immunoblotting for suspected E3 ligase components

  • Implement bidirectional Co-IP (using antibodies against both MRFAP1 and E3 ligase components)

  • Include proteasome inhibitors (MG132) to stabilize transient interactions

  • Consider crosslinking approaches for capturing weak or transient interactions

• Degradation Analysis:

  • Perform cycloheximide chase assays to measure MRFAP1 half-life under various conditions

  • Compare MRFAP1 stability after knockdown or knockout of specific E3 ligase components (FBXW8, CUL4B, CUL7)

  • Use MLN4924 (NEDD8 inhibitor) treatment to block cullin-RING E3 ligase activity and observe MRFAP1 stabilization

• Ubiquitination Assays:

  • Express His-tagged ubiquitin, perform denaturing purification, and analyze MRFAP1 poly-ubiquitination

  • Compare ubiquitination patterns with and without overexpression of E3 ligase components

  • Use in vitro ubiquitination assays with purified components to confirm direct enzymatic activity

Research has established that MRFAP1 interacts with multiple E3 ligases, including Cul7/FBXW8 and CUL4B complexes, with FBXW8 playing a critical role in cell cycle-dependent degradation during anaphase .

How does MRFAP1 expression correlate with cancer progression, and what methodological approaches are recommended for such studies?

MRFAP1 demonstrates altered expression in cancer tissues with potential implications for cancer progression:

• Expression Analysis in Cancer Tissues:

  • Research shows MRFAP1 is downregulated in gastric cancer (GC) tissues at the protein level, despite no significant changes in mRNA levels, indicating post-translational regulation

  • Quantitative analysis using IHC in gastric cancer samples revealed decreased MRFAP1 protein compared to corresponding non-cancerous tissues

• Recommended Methodology for Expression Studies:

  • Implement both qRT-PCR and Western blot analysis on paired cancer/normal tissues to distinguish transcriptional from post-translational regulation

  • Apply IHC on tissue microarrays with appropriate antibody dilutions (1:20-1:200) using suggested antigen retrieval methods

  • For cell line models, compare expression across multiple cancer and non-cancer cell lines using standardized Western blot protocols

• Functional Analysis in Cancer Context:

  • Utilize CRISPR-mediated knockout and overexpression systems to evaluate MRFAP1's impact on cancer cell phenotypes

  • Apply cell proliferation assays (CCK8), cell cycle analysis (flow cytometry), and apoptosis measurements to assess functional consequences

  • Investigate MRFAP1's role in response to therapeutic agents, particularly those targeting the ubiquitin-proteasome system

Research by Hu et al. demonstrated that overexpression of MRFAP1 in gastric cancer cell lines (AGS and SGC-7901) decreased proliferation and induced G1 arrest, suggesting a potential tumor-suppressive role .

What methodological approaches should be employed to investigate MRFAP1's role in response to neddylation inhibitors like MLN4924?

MRFAP1 is one of the most dramatically stabilized proteins following NEDD8 inhibition, suggesting complex roles in neddylation pathways:

• Experimental Design for MLN4924 Studies:

  • Implement dose-response and time-course treatments with MLN4924 across multiple cell lines

  • Combine MRFAP1 knockout or knockdown with MLN4924 treatment to assess functional interactions

  • Apply cell viability, cell cycle, and apoptosis assays to evaluate phenotypic outcomes of combined MRFAP1 modulation and MLN4924 treatment

• Molecular Mechanism Investigation:

  • Analyze the effect of MLN4924 on MRFAP1-MORF4L1 complex formation using co-immunoprecipitation

  • Investigate interactions between MRFAP1 and cell cycle regulators (such as P27) in response to MLN4924

  • Implement proteomics analysis to identify global changes in protein-protein interactions after neddylation inhibition

• Translational Relevance Assessment:

  • Test combination strategies of MRFAP1 inhibition with MLN4924 treatment in cancer models

  • Evaluate synergistic effects on cell growth inhibition, cell cycle arrest, and apoptosis induction

  • Analyze downstream pathway activation using phospho-specific antibodies against key signaling nodes

Recent research demonstrated that CRISPR-mediated knockout of MRFAP1 significantly enhanced the cytotoxicity of MLN4924 in gastric cancer cells by augmenting G2/M arrest and apoptosis, suggesting potential combinatorial therapeutic approaches .

What are the critical parameters for optimizing MRFAP1 antibody-based Western blot protocols?

Optimizing Western blot protocols for MRFAP1 detection requires attention to several critical parameters:

• Sample Preparation Considerations:

  • Include proteasome inhibitors (MG132) in lysis buffers to prevent rapid degradation of MRFAP1

  • Consider cell cycle stage when harvesting cells due to MRFAP1's cell cycle-dependent expression

  • Implement phosphatase inhibitors to preserve potential post-translational modifications

  • Use denaturing conditions with SDS and reducing agents to ensure complete protein denaturation

• Electrophoresis and Transfer Parameters:

  • Optimize gel percentage (12-15%) for optimal resolution of the 15 kDa MRFAP1 protein

  • Consider gradient gels when analyzing both MRFAP1 and its interaction partners

  • Implement semi-dry or wet transfer methods optimized for small proteins

  • Use PVDF membranes with 0.2 μm pore size for improved retention of small proteins

• Antibody Incubation and Detection:

  • Titrate primary antibody concentration within the recommended range (1:200-1:2000)

  • Test both short (1-2 hour) and overnight primary antibody incubations at different temperatures

  • Implement enhanced chemiluminescence detection systems suitable for low-abundance proteins

  • Consider fluorescent secondary antibodies for multiplex detection with cell cycle markers

For validation, researchers should include positive control samples (such as Jurkat cell lysates) and compare results with published molecular weight observations (15 kDa) .

How should researchers interpret contradictory findings when using MRFAP1 antibodies across different experimental systems?

When faced with contradictory results using MRFAP1 antibodies, researchers should implement a systematic troubleshooting approach:

• Antibody Validation Assessment:

  • Verify antibody specificity using MRFAP1 knockout or knockdown controls

  • Test multiple antibodies targeting different epitopes of MRFAP1

  • Compare results from monoclonal versus polyclonal antibodies

  • Confirm antibody reactivity with both endogenous and overexpressed MRFAP1

• Biological Variability Analysis:

  • Consider cell type-specific expression patterns (MRFAP1 shows tissue-specific expression)

  • Evaluate cell cycle stage distribution in your experimental system (MRFAP1 levels fluctuate during mitosis)

  • Assess potential post-translational modifications affecting epitope recognition

  • Analyze mRNA expression in parallel to distinguish transcriptional from post-translational effects

• Methodological Reconciliation:

  • Compare detection methods (WB, IHC, IF) for consistent or divergent results

  • Standardize sample preparation protocols across experimental systems

  • Implement quantitative approaches (densitometry, digital pathology) for objective comparison

  • Consider the impact of fixation methods on epitope availability in IHC/IF applications

Research demonstrates that MRFAP1 expression can vary dramatically based on cell cycle stage, with rapid degradation during anaphase, which could lead to apparent contradictions depending on cell synchronization status .

What are the optimal storage conditions for maintaining MRFAP1 antibody activity, and how should researchers validate antibody performance over time?

Proper storage and validation of MRFAP1 antibodies are critical for experimental reproducibility:

• Optimal Storage Parameters:

  • Store MRFAP1 antibodies at -20°C in manufacturer-recommended buffer systems (typically PBS with 0.02% sodium azide and 50% glycerol, pH 7.3)

  • Avoid repeated freeze-thaw cycles by preparing small working aliquots

  • Most antibodies remain stable for one year after shipment when properly stored

  • For short-term storage of working solutions, maintain at 4°C for no longer than one week

• Performance Validation Methodology:

  • Implement routine validation using positive control samples (e.g., Jurkat cell lysates)

  • Maintain a reference lot for comparison when obtaining new antibody batches

  • Document lot-to-lot variation by comparing signal intensity and specificity

  • Track antibody performance over time with standardized protocols and consistent detection methods

• Quality Control Measures:

  • Verify antibody concentration periodically using spectrophotometric methods

  • Test for contamination by running antibody-only controls

  • Implement periodical specificity testing using knockdown/knockout controls

  • Consider preparing standard curves with recombinant MRFAP1 protein for quantitative applications

Researchers should note that antibody recycling is generally not recommended for MRFAP1 detection, as buffer systems change after use and storage conditions of recycled antibodies can vary, affecting performance reliability .

How can researchers distinguish between true MRFAP1 signals and nonspecific binding in complex experimental systems?

Distinguishing specific from nonspecific signals is particularly important for MRFAP1 detection:

• Control Implementation Strategy:

  • Generate and utilize MRFAP1 knockout cell lines via CRISPR/Cas9 as negative controls

  • Implement siRNA-mediated knockdown of MRFAP1 with scrambled siRNA controls

  • Include competitive blocking with immunizing peptides to confirm specificity

  • Apply isotype control antibodies to identify Fc receptor-mediated background

• Technical Verification Approaches:

  • Compare signal patterns across multiple MRFAP1 antibodies targeting different epitopes

  • Verify molecular weight precision (15 kDa) in Western blot applications

  • Confirm expected subcellular localization patterns (nuclear) in immunofluorescence

  • Validate tissue expression patterns with established MRFAP1 distribution profiles (highest in testis and brain)

• Signal Validation Methodology:

  • Implement titration experiments to determine optimal antibody concentration

  • Compare native versus denatured detection systems to identify conformation-dependent signals

  • Apply orthogonal detection methods (MS-based proteomics) to confirm MRFAP1 presence

  • Use recombinant MRFAP1 protein for antibody pre-absorption tests

Research demonstrates that MRFAP1 shows specific expression patterns, with highest levels in spermatogonia within seminiferous tubules of testis and much weaker staining in spermatocytes and spermatids—such known distribution patterns can serve as biological validation points .