MPHOSPH10 Antibody

What is MPHOSPH10 and why is it significant in cellular research?

MPHOSPH10 (M-Phase phosphoprotein 10) is a protein associated with the U3 small nucleolar ribonucleoprotein complex. It holds significant research interest because it exhibits dynamic localization patterns throughout the cell cycle, residing in the nucleolus during interphase and relocating to chromosomes during M phase. MPHOSPH10 becomes phosphorylated during mitosis and is implicated in pre-rRNA processing through its association with U3 small nucleolar ribonucleoprotein 60-80S complexes . The protein's involvement in nucleolar function makes it an important target for studying ribosome biogenesis and cell cycle regulation. Recent nucleolar proteome mapping has identified MPHOSPH10 as part of a larger-than-previously-thought nucleolar protein network, suggesting additional functional roles that remain to be fully characterized .

What types of MPHOSPH10 antibodies are available for research applications?

Multiple types of MPHOSPH10 antibodies are available to researchers, varying in host species, clonality, target epitopes, and applications:

These varied antibodies enable researchers to target different epitopes of MPHOSPH10, which can be particularly valuable when investigating protein interactions, post-translational modifications, or when validating results using multiple independent antibodies .

What are the validated applications for MPHOSPH10 antibodies?

MPHOSPH10 antibodies have been validated for several research applications with specific optimal dilutions and conditions:

• Western Blotting (WB): Most commercially available MPHOSPH10 antibodies are validated for WB, typically at dilutions of 1:500-1:1000 . The expected molecular weight is approximately 78 kDa, though observed band sizes may vary due to post-translational modifications or splice variants .

• Immunohistochemistry (IHC): Paraffin-embedded tissue sections can be analyzed using dilutions of 1:200-1:500 for optimal staining .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Validated protocols exist using 0.25-2 μg/ml concentrations for cellular localization studies .

• ELISA: Several antibodies have been validated for enzyme-linked immunosorbent assays, though specific protocols vary by manufacturer .

• Immunoprecipitation (IP): Some rabbit polyclonal antibodies have been validated for pulling down native MPHOSPH10 protein complexes .

It's important to note that each antibody may have different optimal conditions for specific applications, and researchers should perform validation tests before proceeding with critical experiments .

How should researchers select the appropriate MPHOSPH10 antibody for their specific research question?

Selection of the appropriate MPHOSPH10 antibody should be guided by several experimental considerations:

• Target species: Verify cross-reactivity with your experimental model. Some antibodies react only with human MPHOSPH10, while others are validated for mouse, rat, and even non-mammalian models like zebrafish .

• Application compatibility: Choose antibodies specifically validated for your intended application. For example, if studying protein localization during mitosis, select antibodies validated for immunofluorescence with demonstrated specificity for phosphorylated forms .

• Epitope location: Consider whether the protein region of interest might be masked by interactions or modifications. C-terminal antibodies may be preferable when studying full-length protein, while internal region antibodies may be better for detecting specific domains or truncated forms .

• Validation status: Prioritize antibodies with higher validation scores. Some providers use scoring systems (enhanced, supported, approved, or uncertain) based on additional validation methods like siRNA knockdown or consistency between independent antibodies .

• Clonality needs: For exploratory studies, polyclonal antibodies offer broader epitope recognition, while monoclonal antibodies provide greater specificity and reproducibility for targeted analyses .

How can researchers optimize Western blotting protocols for MPHOSPH10 detection?

Optimizing Western blotting for MPHOSPH10 requires careful attention to several key parameters:

• Sample preparation: Since MPHOSPH10 is a nucleolar protein that relocates during mitosis, optimal extraction requires thorough nuclear lysis. Use buffers containing 1% Triton X-100 or RIPA buffer supplemented with benzonase to ensure complete chromatin solubilization .

• Running conditions: MPHOSPH10's calculated molecular weight is 78 kDa, but observed molecular weights may vary due to post-translational modifications. Use 8-10% polyacrylamide gels with extended running times to achieve optimal separation .

• Transfer parameters: For proteins of this size, semi-dry transfer systems may be insufficient. Use wet transfer at 30V overnight at 4°C with methanol-free transfer buffer containing 0.1% SDS to improve transfer efficiency .

• Blocking and antibody dilutions: Most validated protocols recommend 5% non-fat milk in TBST for blocking, with primary antibody dilutions of 1:500-1:1000 . Overnight incubation at 4°C is typically optimal for signal development.

• Detection method selection: For phosphorylation studies, highly sensitive chemiluminescent substrates are recommended due to the transient nature of phosphorylation during the cell cycle .

When troubleshooting weak signals, researchers should consider cell synchronization to enrich for mitotic cells where MPHOSPH10 is phosphorylated, potentially enhancing detection of specific modified forms .

What methodological approaches can verify MPHOSPH10 subcellular localization throughout the cell cycle?

Verifying MPHOSPH10's dynamic subcellular localization requires carefully designed experimental approaches:

• Cell synchronization: To capture different cell cycle phases, use double thymidine block (G1/S boundary), nocodazole treatment (M phase), or serum starvation followed by release (G0/G1) .

• Co-localization markers: Pair MPHOSPH10 antibodies with established markers such as fibrillarin (nucleolar), phospho-histone H3 (mitotic chromosomes), and BrdU incorporation (S phase) .

• Live-cell imaging: For dynamic studies, consider generating stable cell lines expressing MPHOSPH10-GFP fusion proteins, validated against antibody staining patterns to ensure proper localization .

• Super-resolution microscopy: Techniques like structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) provide enhanced resolution for precise localization within nucleolar subcompartments .

• Biochemical fractionation: Complement imaging data with subcellular fractionation followed by Western blotting to quantify MPHOSPH10 distribution across nucleolar, nucleoplasmic, and chromosomal fractions throughout the cell cycle .

A robust verification approach should combine at least two independent methods, such as immunofluorescence with fractionation biochemistry, to conclusively establish localization patterns and avoid technique-specific artifacts .

How do post-translational modifications affect MPHOSPH10 antibody recognition?

Post-translational modifications (PTMs) of MPHOSPH10 can significantly impact antibody recognition in several ways:

• Phosphorylation effects: MPHOSPH10 becomes phosphorylated during mitosis, which may expose or mask epitopes recognized by certain antibodies . Phospho-specific antibodies may only recognize the modified form, while some antibodies may show reduced binding to phosphorylated protein.

• Epitope accessibility: When using antibodies targeting specific regions, researchers should consider whether PTMs in proximity to the epitope might interfere with binding. C-terminal antibodies may be particularly sensitive to modifications in that region .

• Conformation changes: PTMs can induce conformational changes that alter antibody accessibility to linear epitopes. Internal region antibodies may be more susceptible to these effects than terminal-targeting antibodies .

• Technical considerations: When studying phosphorylated MPHOSPH10, samples should be prepared with phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate) to preserve modification status .

• Validation approach: To distinguish between PTM-dependent recognition patterns, researchers can compare signal patterns before and after treating lysates with phosphatases, or use synchronized cell populations enriched for specific phosphorylation states .

The observed molecular weight of MPHOSPH10 (78 kDa) may show band shifts on Western blots due to these modifications, and multiple bands may indicate the presence of different modified forms within the same sample .

What experimental approaches can verify MPHOSPH10 interactions with the U3 snoRNP complex?

Verifying MPHOSPH10's interactions with the U3 snoRNP complex requires multiple complementary approaches:

• Co-immunoprecipitation (Co-IP): Use MPHOSPH10 antibodies for IP followed by Western blotting for known U3 snoRNP components (fibrillarin, NOP58, NOP56, UTP proteins) . Alternatively, perform reverse Co-IP using antibodies against established U3 snoRNP components.

• Proximity ligation assay (PLA): This technique can visualize protein-protein interactions in situ with high sensitivity. Pair MPHOSPH10 antibodies with antibodies against U3 snoRNP components to generate fluorescent signals only when proteins are in close proximity (<40 nm) .

• RNA immunoprecipitation (RIP): To confirm association with U3 snoRNA, perform RIP using MPHOSPH10 antibodies followed by RT-PCR or Northern blotting for U3 snoRNA .

• Mass spectrometry validation: Immunoprecipitate MPHOSPH10 under native conditions and analyze protein complexes by mass spectrometry to identify all associated proteins and confirm U3 snoRNP components .

• Functional reconstitution: For definitive proof of functional association, deplete endogenous MPHOSPH10 using siRNA and rescue with recombinant protein to assess restoration of U3 snoRNP assembly and function .

When designing these experiments, researchers should consider cell synchronization to capture specific cell cycle phases, as MPHOSPH10's interactions may be dynamic and phase-dependent .

What cell lines are most suitable for studying MPHOSPH10 expression and function?

Selection of appropriate cell lines for MPHOSPH10 research depends on specific research objectives:

• Expression level considerations: MPHOSPH10 expression varies across cell types. Based on nucleolar proteome studies, cell lines with prominent nucleoli such as HeLa, U2OS, and MCF7 express detectable levels of MPHOSPH10 .

• Cell cycle research: For cell cycle studies, choose cell lines with well-characterized and relatively synchronous cell cycles, such as HeLa or RPE-1 cells, which can be effectively synchronized using standard protocols .

• Species-specific studies: For cross-species comparisons, select antibodies with validated cross-reactivity. Some MPHOSPH10 antibodies recognize human, mouse, rat, dog, horse, rabbit, zebrafish, and monkey orthologs .

• Nucleolar function research: Cell lines with large, prominent nucleoli (e.g., cancer cell lines like HeLa) are advantageous for studying MPHOSPH10's nucleolar functions and pre-rRNA processing roles .

• Technical considerations: For immunocytochemistry/immunofluorescence applications, adherent cell lines that maintain flat morphology during fixation (U2OS, MCF7) provide superior imaging quality compared to cells with rounded morphology .

Researchers should validate MPHOSPH10 expression in their chosen cell line using Western blotting before proceeding with functional studies, as expression levels and patterns may vary between even closely related cell types .

How can researchers study MPHOSPH10's role in pre-rRNA processing?

Investigating MPHOSPH10's role in pre-rRNA processing requires specialized approaches focused on ribosome biogenesis:

• RNA interference: Deplete MPHOSPH10 using siRNA or shRNA approaches, followed by Northern blot analysis of pre-rRNA processing intermediates using probes targeting specific regions of the 47S pre-rRNA transcript .

• Pulse-chase labeling: Use 5-ethynyl uridine (EU) or methionine pulse labeling followed by chase periods to track nascent rRNA synthesis and processing rates in the presence or absence of MPHOSPH10 .

• RNA-protein interaction mapping: Employ CLIP-seq (Cross-linking immunoprecipitation followed by sequencing) using MPHOSPH10 antibodies to identify direct RNA binding sites within pre-rRNA transcripts .

• Structure-function analysis: Generate domain deletion or point mutation constructs of MPHOSPH10 to identify regions required for pre-rRNA processing, followed by rescue experiments in MPHOSPH10-depleted cells .

• Metabolic labeling: Use 32P-orthophosphate labeling to track newly synthesized rRNA in MPHOSPH10-depleted versus control cells, allowing quantitative assessment of processing defects .

• Polysome profiling: Analyze polysome profiles to assess the impact of MPHOSPH10 depletion on mature ribosome formation, which would be affected by defects in pre-rRNA processing .

When interpreting results, researchers should consider that severe defects in pre-rRNA processing often trigger nucleolar stress responses that can complicate analysis, necessitating careful time-course studies to distinguish primary from secondary effects .

How should researchers validate a new MPHOSPH10 antibody for their research?

Comprehensive validation of a new MPHOSPH10 antibody should follow these methodological steps:

• Positive and negative controls: Test the antibody on samples with known MPHOSPH10 expression (positive control) and on samples where MPHOSPH10 has been depleted through siRNA/shRNA knockdown (negative control) .

• Cross-reactivity assessment: For antibodies claiming multi-species reactivity, validate each species individually rather than assuming conserved reactivity. Test tissues or cell lines from human, mouse, rat, and other relevant species .

• Multiple application validation: Even if an antibody is validated for one application (e.g., Western blotting), independently validate it for each intended use (IHC, ICC, IP) .

• Epitope blocking: Perform peptide competition assays using the immunizing peptide to confirm epitope specificity. Signal abolishment indicates specific binding .

• Orthogonal validation: Compare results with independent antibodies targeting different epitopes of MPHOSPH10, or with non-antibody methods like GFP-tagging or mass spectrometry .

• Localization consistency: For immunofluorescence applications, verify that the observed localization pattern matches the expected nucleolar localization during interphase and chromosomal association during mitosis .

• Band size verification: For Western blotting, confirm that the detected band matches the expected molecular weight of 78 kDa, while being aware that post-translational modifications may cause shifts in apparent molecular weight .

Documentation of validation experiments is essential for publication and reproducibility, with images of positive and negative controls included in supplementary materials .

What are common pitfalls when using MPHOSPH10 antibodies and how can they be addressed?

Researchers should be aware of several common pitfalls when working with MPHOSPH10 antibodies:

Proper controls and validation steps at each experimental stage can mitigate these potential problems and ensure reliable results .

How can researchers address non-specific binding with MPHOSPH10 antibodies?

Non-specific binding is a common challenge with MPHOSPH10 antibodies that can be systematically addressed:

• Optimization of blocking conditions: Test different blocking agents (5% BSA, 5% non-fat milk, commercial blocking buffers) to identify optimal conditions that minimize background while preserving specific signal .

• Antibody titration: Perform dilution series experiments to determine the minimum antibody concentration that provides specific signal. Using excess antibody often increases non-specific binding .

• Pre-adsorption strategies: For tissues with high background, pre-adsorb the antibody against acetone powder prepared from negative control tissues to remove cross-reactive antibodies .

• Buffer optimization: Increasing salt concentration (up to 500 mM NaCl) or adding mild detergents (0.1% Triton X-100) to wash buffers can reduce hydrophobic non-specific interactions .

• Secondary antibody controls: Always include controls omitting primary antibody to identify non-specific binding from secondary antibodies, particularly in highly autofluorescent tissues .

• Cross-adsorbed secondary antibodies: Use highly cross-adsorbed secondary antibodies to prevent recognition of endogenous immunoglobulins, especially important for tissue immunohistochemistry .

• Peptide competition: Perform peptide competition assays by pre-incubating the antibody with excess immunizing peptide. Specific signals should be blocked while non-specific signals remain .

• Orthogonal validation: Compare staining patterns between multiple independent MPHOSPH10 antibodies targeting different epitopes. Consistent patterns across antibodies suggest specific binding .

When publishing, researchers should document optimization steps taken to address non-specific binding, including representative images of control experiments .

How does MPHOSPH10 contribute to the expanded understanding of the nucleolar proteome?

Recent advances in nucleolar proteome mapping have positioned MPHOSPH10 within a broader functional context:

• Expanded nucleolar protein catalog: Comprehensive studies have revealed that the nucleolar proteome is larger than previously thought, with MPHOSPH10 being one of the proteins confirmed through multiple independent approaches .

• Spatial organization insights: MPHOSPH10's localization patterns contribute to understanding the spatiotemporal organization of nucleolar functions, particularly how proteins redistribute between nucleolar compartments during different cellular states .

• Integration with network analysis: MPHOSPH10's interactions with the U3 snoRNP complex place it within a functional network involved in pre-rRNA processing, helping researchers map the complete pathway of ribosome biogenesis .

• Cell type-specific variations: Studies across multiple cell lines have revealed that while MPHOSPH10 is broadly expressed, its relative abundance and interaction partners may vary between cell types, contributing to our understanding of tissue-specific nucleolar functions .

• Technical advances in detection: The development of specific antibodies targeting different MPHOSPH10 epitopes has enabled more precise localization studies, revealing previously unrecognized patterns of distribution within nucleolar subcompartments .

Researchers leveraging these insights should consider MPHOSPH10 not in isolation but as part of an integrated nucleolar functional network that dynamically responds to cellular conditions and stresses .

What are the current technical limitations in MPHOSPH10 research?

Despite significant advances, several technical limitations persist in MPHOSPH10 research:

• Temporal resolution challenges: Standard immunofluorescence provides static snapshots of MPHOSPH10 localization, limiting understanding of its dynamic behavior. Advanced live-cell imaging approaches with minimal photobleaching are needed .

• Epitope masking during complex formation: MPHOSPH10's incorporation into large ribonucleoprotein complexes may mask epitopes, potentially leading to underestimation of true protein levels or false-negative results in certain applications .

• Post-translational modification detection: Current antibodies primarily detect total MPHOSPH10 rather than specific phosphorylated forms. Development of modification-specific antibodies is needed to fully characterize its cell cycle-dependent regulation .

• Cross-reactivity limitations: While some antibodies claim cross-reactivity across multiple species, systematic validation across evolutionary diverse models remains limited, constraining comparative studies .

• Functional redundancy complications: Potential functional redundancy with other nucleolar proteins complicates knockdown studies, as compensatory mechanisms may mask phenotypes. Acute depletion systems like auxin-inducible degrons might address this limitation .

• Structural information gaps: The absence of crystal structures for MPHOSPH10 limits rational design of function-blocking antibodies or small molecule inhibitors that could provide more precise tools for functional studies .

How can researchers contribute to improving MPHOSPH10 antibody resources?

The scientific community can systematically improve MPHOSPH10 antibody resources through collaborative efforts:

• Independent validation reporting: Researchers who validate MPHOSPH10 antibodies for specific applications should publish detailed protocols and validation data, including negative controls and application-specific optimization parameters .

• Antibody registry contributions: Submit validation data to community resources like Antibodypedia or the Antibody Registry to help other researchers select appropriate reagents for their specific applications .

• Development of recombinant antibodies: Consider converting valuable hybridoma-derived antibodies to recombinant formats to ensure long-term reproducibility and eliminate batch-to-batch variation inherent to polyclonal resources .

• Modification-specific antibodies: Develop and characterize antibodies specific to phosphorylated forms of MPHOSPH10 to enable more precise studies of its cell cycle-dependent regulation .

• Cross-laboratory validation initiatives: Participate in multi-laboratory validation studies to assess reproducibility across different research environments, strengthening confidence in antibody specificity .

• Open science practices: Share detailed immunogen information, validation protocols, and negative results through repositories like protocols.io or the Resource Identification Portal to reduce redundant validation efforts .

• Application expansion: Validate existing antibodies for emerging techniques like proximity labeling, ChIP-seq, or super-resolution microscopy to expand the methodological toolkit available to the MPHOSPH10 research community .