MYBBP1A Antibody

What is MYBBP1A and what cellular functions make it an important research target?

MYBBP1A (also known as P160) is a 149 kDa nucleolar protein with multiple critical cellular functions. It serves as a transcriptional regulator that can either activate or repress transcription through interactions with sequence-specific DNA-binding proteins . MYBBP1A acts as a corepressor and, in concert with CRY1, represses transcription of the core circadian clock component PER2 .

Research has established multiple critical functions of MYBBP1A:

• Regulation of RNA polymerase I transcription and pre-rRNA processing

• Enhancement of p53 tetramerization during nucleolar stress

• Tumor suppression in breast cancer and hepatocellular carcinoma

• Epigenetic silencing of myogenic gene programs

• Coordination of rRNA gene transcription and processing

These diverse functions make MYBBP1A antibodies essential tools for researchers investigating nucleolar function, transcriptional regulation, and cancer biology.

What applications are validated for MYBBP1A antibodies?

Based on extensive validation data from multiple manufacturers, MYBBP1A antibodies have been successfully employed in various experimental applications:

When selecting an antibody for a specific application, researchers should review the validation data provided by manufacturers for that particular application .

What dilutions and experimental conditions are recommended for different applications?

Optimal dilutions vary by application and specific antibody. The following table provides a general range of recommended dilutions based on multiple commercial antibodies:

For IHC applications with FFPE tissue sections, epitope retrieval is recommended:

• Citrate buffer (pH 6.0) or TE buffer (pH 9.0) for optimal antigen retrieval

• Validated positive controls include human kidney tissue and human colon carcinoma

It's critical to note that optimal concentrations should be determined experimentally for each specific system and application .

How can researchers effectively study MYBBP1A's role in p53 activation and cellular stress response?

MYBBP1A plays a crucial role in p53 activation during nucleolar stress. Researchers investigating this pathway should consider the following methodological approaches:

Experimental approach for studying MYBBP1A-p53 interaction:

• Subcellular localization studies: Track MYBBP1A translocation from nucleolus to nucleoplasm under stress conditions using immunofluorescence with validated antibodies

• Co-immunoprecipitation assays: Use sequential Co-IP to detect MYBBP1A-p53 interactions that form during stress conditions

• Tetramerization analysis: Employ electrophoretic mobility shift assay (EMSA) to assess p53 tetramerization enhancement by MYBBP1A

• Acetylation detection: Monitor p53 K382 acetylation levels as a downstream indicator of MYBBP1A-mediated p53 activation

• ChIP assays: Measure p53 recruitment to target promoters (e.g., Bax) to assess MYBBP1A's impact on p53 transcriptional activity

When conducting nucleolar stress experiments, researchers have successfully used actinomycin D treatment to induce stress and measure MYBBP1A-dependent p53 activation .

What are the key considerations when investigating MYBBP1A's tumor suppressor function?

MYBBP1A has been established as a tumor suppressor in multiple cancer types, including breast cancer and hepatocellular carcinoma. Researchers investigating this role should consider:

Methodological approaches:

• Expression analysis in clinical samples: Correlate MYBBP1A expression with cancer progression using patient microarray databases and tissue microarrays

• Knockdown and overexpression models: Generate stable cell lines with MYBBP1A knockdown or overexpression to study functional effects

• Colony formation assays: Assess ability of cells to form colonies in soft agar with modulated MYBBP1A expression

• Xenograft models: Evaluate tumorigenicity in vivo using cells with altered MYBBP1A expression

• Anoikis assays: Test resistance to anoikis (detachment-induced apoptosis) as a key mechanism in MYBBP1A's tumor suppressor function

Research has shown MYBBP1A suppresses HCC progression through inhibiting the IGF1/AKT signaling pathway by forming a complex with DNMT1 and inducing hypermethylation of IGFBP5 CpG islands .

How can researchers validate the specificity of MYBBP1A antibodies?

Ensuring antibody specificity is crucial for reliable results. Multiple validation approaches should be considered:

• Positive and negative controls:

  • Use cell lines with confirmed MYBBP1A expression (validated positive controls include HeLa, HEK-293, SW620, and MCF7 cells)

  • Employ MYBBP1A knockdown/knockout cells as negative controls

• Molecular weight verification: Confirm detection of the expected molecular weight (149-160 kDa) in Western blots

• Cross-validation with multiple antibodies: Compare results with at least two independent MYBBP1A antibodies targeting different epitopes

• Peptide competition assays: Perform blocking experiments with the immunizing peptide to confirm specificity

• Immunoprecipitation validation: Verify ability to immunoprecipitate MYBBP1A and confirm by Western blot with a different MYBBP1A antibody

The scientific literature contains published validation using MYBBP1A antibodies in KD/KO systems, providing strong reference points for validation strategies .

What are the most common technical challenges when detecting MYBBP1A in different cellular compartments?

MYBBP1A primarily localizes to the nucleolus but translocates to the nucleoplasm under various stress conditions. This dynamic localization presents several technical challenges:

Nucleolar detection challenges:

• Nucleolar proteins often require specialized fixation protocols to maintain structure integrity

• Nuclear extraction protocols may result in incomplete nucleolar protein recovery

• Competitive binding within dense nucleolar structures may impede antibody access

Solutions and approaches:

• Optimized fixation protocols: For IF/ICC applications, test multiple fixation methods (4% paraformaldehyde, methanol/acetone) to determine optimal conditions for nucleolar structure preservation

• Pre-extraction steps: Employ pre-extraction with Triton X-100 before fixation to improve antibody accessibility to nucleolar structures

• Sequential extraction: Use sequential nuclear extraction protocols specifically designed for nucleolar proteins

• Subcellular fractionation: For biochemical analysis, employ protocols that specifically separate nucleolar, nucleoplasmic, and cytoplasmic fractions

When studying MYBBP1A translocation during stress response, carefully titrate stress inducers (like actinomycin D) and optimize timepoints for capturing the dynamic process .

How can researchers effectively study MYBBP1A's role in regulating rRNA transcription and processing?

MYBBP1A plays a complex dual role in regulating both rRNA gene transcription and pre-rRNA processing. Researchers investigating these processes should consider:

Experimental approaches:

• Reporter assays: Employ reporter systems that uncouple transcription and RNA processing to study MYBBP1A's direct effect on rRNA gene transcription

• ChIP assays: Use chromatin immunoprecipitation to assess RNA polymerase I loading on rRNA genes in the presence/absence of MYBBP1A

• RNA analysis: Monitor accumulation of rRNA precursors using Northern blotting or quantitative RT-PCR to detect processing defects

• Protein complex analysis: Investigate MYBBP1A association with both RNA polymerase I complexes and ribosome biogenesis machinery using immunoprecipitation followed by mass spectrometry

Research has demonstrated that MYBBP1A represses rRNA gene transcription while simultaneously being required for proper pre-rRNA processing, suggesting a coordinating role between these processes .

What are the key considerations for analyzing MYBBP1A interactions with other proteins using co-immunoprecipitation?

MYBBP1A participates in multiple protein-protein interactions that are crucial for its various functions. When performing co-immunoprecipitation studies:

• Antibody selection: Choose antibodies validated for immunoprecipitation applications

• Lysis conditions: Test multiple lysis buffers (NETN, RIPA, etc.) as buffer composition significantly affects protein complex stability

• Sequential Co-IP: For complex protein interactions, employ sequential Co-IP to verify multi-protein complexes

• Controls: Include appropriate controls (IgG control, reversed Co-IP, competition with immunizing peptide)

• Cross-linking: Consider mild cross-linking approaches for capturing transient interactions

• Nuclear extraction: Use optimized nuclear extraction protocols as MYBBP1A primarily localizes to the nucleus/nucleolus

For studying MYBBP1A-p53 interactions specifically, research shows these interactions form primarily under nucleolar stress conditions, so experimental design should include appropriate stress induction .

How can MYBBP1A antibodies be used to study its role in cancer progression?

MYBBP1A serves as a tumor suppressor in multiple cancer types. Researchers studying its role in oncology should consider:

Research applications:

• Expression analysis in clinical samples: Use IHC with validated MYBBP1A antibodies to correlate expression levels with clinical parameters and patient outcomes

• Mechanistic studies: Employ IP and ChIP approaches to determine how MYBBP1A regulates key oncogenic pathways

• Epigenetic regulation: Investigate MYBBP1A's role in the epigenetic silencing of genes by analyzing its interaction with chromatin modifiers using Co-IP and ChIP approaches

• Signaling pathway analysis: Study MYBBP1A's effect on critical signaling pathways (e.g., IGF1/AKT in HCC) using phospho-specific antibodies in combination with MYBBP1A modulation

Research has demonstrated that MYBBP1A expression is negatively correlated with breast cancer tumorigenesis, and its knockdown enhances colony formation, tumorigenesis, and anoikis resistance in breast cancer cell lines .

What are the experimental considerations when studying MYBBP1A-mediated regulation of gene expression?

MYBBP1A functions as a transcriptional coregulator through multiple mechanisms. When investigating these activities:

• ChIP-seq approaches: Employ chromatin immunoprecipitation followed by sequencing to identify genome-wide binding sites of MYBBP1A

• Transcriptional reporter assays: Use reporter systems to measure MYBBP1A's direct effect on the transcriptional activity of specific promoters

• mRNA expression analysis: Compare gene expression profiles in MYBBP1A-depleted versus control cells to identify regulated targets

• Co-repressor complex analysis: Investigate MYBBP1A's recruitment of histone deacetylases (HDACs) and other epigenetic modifiers to specific promoters

• DNA methylation analysis: Examine MYBBP1A's role in DNA methylation by studying its interaction with DNA methyltransferases and analyzing methylation patterns at target genes

Research has shown that MYBBP1A exerts its repressive role by inducing a less permissive chromatin structure following recruitment of negative epigenetic modifiers .

How is MYBBP1A being investigated as a potential biomarker or therapeutic target?

Recent research has highlighted MYBBP1A's potential as both a biomarker and therapeutic target:

• Biomarker applications:

  • Expression analysis in tumor tissues for prognostic evaluation

  • Correlation with treatment response in various cancer types

  • Assessment of nucleolar stress response activation

• Therapeutic targeting strategies:

  • Modulation of MYBBP1A expression to enhance p53-dependent tumor suppression

  • Targeting MYBBP1A-dependent epigenetic regulation mechanisms

  • Developing approaches to regulate MYBBP1A's nucleolar-nucleoplasmic shuttling

Research has identified that loss of MYBBP1A occurs in 8-9% of renal tumors and may occur in other tumor types, suggesting potential as a biomarker for specific cancer subpopulations that could be targeted with novel therapeutic approaches .

What methodological advances are improving the study of MYBBP1A functions?

Several technological and methodological advances are enhancing MYBBP1A research:

• Proximity labeling approaches: BioID and APEX2-based approaches to identify proximal proteins in different subcellular compartments

• CRISPR-Cas9 genome editing: Generation of precise knockout and knock-in cell lines for studying MYBBP1A function

• Super-resolution microscopy: Improved visualization of MYBBP1A's subnucleolar localization and dynamics

• Single-cell approaches: Analysis of MYBBP1A expression and function at the single-cell level to understand cellular heterogeneity

• Proteomics integration: Combination of antibody-based detection with mass spectrometry for comprehensive protein interaction studies

The development of increasingly specific antibodies with validation across multiple applications has significantly advanced researchers' ability to study MYBBP1A's diverse functions across different experimental contexts .