MPHOSPH6 Antibody

What is MPHOSPH6 and what experimental approaches are most effective for studying its cellular functions?

MPHOSPH6 (M-phase phosphoprotein 6) is a 160 amino acid protein with an observed molecular weight of 19 kDa that plays crucial roles in cell cycle regulation, particularly during the transition from G2 to M phase. It belongs to the MPP6 family and contains a nuclear localization signal motif .

The most effective experimental approaches include:

• RNA processing studies: MPHOSPH6 functions in RNA binding (preferentially to pyrimidine homopolymers) and is important for the maturation of 5.8S rRNA, acting in the recruitment of the exosome to pre-rRNA .

• Cell cycle synchronization experiments: Since MPHOSPH6 is phosphorylated during M phase (either directly by MPF or indirectly by kinases activated by MPF), synchronized cell populations can reveal its dynamic regulation .

• Cellular localization experiments: Immunofluorescence with MPHOSPH6 antibodies can track its nuclear localization and redistribution during cell cycle progression.

Validated cell lines for MPHOSPH6 expression include MCF7, HeLa, MCF-7, and MDA-MB-453s, which have shown positive Western blot detection .

What are the validated applications for MPHOSPH6 antibodies and how do their performance characteristics differ?

MPHOSPH6 antibodies have been validated for multiple applications with specific performance parameters:

Different antibody formats show varying characteristics:

• Monoclonal antibodies (like clone A-9 and 36-Y) demonstrate high specificity and reproducibility for targeted epitopes .

• Polyclonal antibodies provide broader epitope recognition, potentially enhancing sensitivity in certain applications .

For optimal results, researchers should titrate antibodies in each specific testing system as performance can be sample-dependent .

What are the critical parameters for troubleshooting Western blot analyses with MPHOSPH6 antibodies?

When troubleshooting Western blots using MPHOSPH6 antibodies, researchers should consider:

• Protein extraction optimization:

  • Nuclear protein extraction is critical as MPHOSPH6 is predominantly nuclear-localized

  • Ensure phosphatase inhibitors are included in lysis buffers, as MPHOSPH6 is a phosphoprotein

• Antibody selection considerations:

  • For human samples, multiple antibodies show reactivity with the expected 19 kDa band

  • For mouse/rat samples, carefully select antibodies validated for cross-reactivity

• Blocking optimization:

  • When using mouse monoclonal antibodies (like clone A-9), block with 5% non-fat milk or BSA

  • When using secondary detection systems, pre-block with species-specific serum to reduce background

• Signal enhancement strategies:

  • Consider using concentrated samples (10-30 μg total protein)

  • Extended primary antibody incubation (overnight at 4°C) may improve detection

  • HRP-conjugated detection reagents generally provide sufficient sensitivity

If unexpected bands appear, verify specificity using positive controls from validated cell lines (MCF7, HeLa) .

How should researchers optimize MPHOSPH6 antibody protocols for immunohistochemistry applications?

For optimal immunohistochemistry results with MPHOSPH6 antibodies:

• Antigen retrieval optimization:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

• Antibody titration strategy:

  • Begin with manufacturer-recommended dilutions (1:20-1:200 for IHC)

  • Perform sequential titrations to determine optimal signal-to-noise ratio

  • Test multiple antibody incubation times (1 hour at RT vs. overnight at 4°C)

• Detection system selection:

  • For routine detection: Standard HRP/DAB systems

  • For co-localization studies: Consider fluorescent secondary antibodies

  • For enhanced sensitivity: Biotin-streptavidin amplification systems

• Validation controls:

  • Positive tissue control: Human breast cancer tissue has been validated

  • Negative controls: Both primary antibody omission and isotype controls

  • Blocking peptide competition: Several manufacturers offer matching blocking peptides

Researchers should note that MPHOSPH6 staining patterns are predominantly nuclear, with potential cell cycle-dependent intensity variations .

What strategies exist for validating the specificity of MPHOSPH6 antibodies in experimental systems?

Comprehensive validation of MPHOSPH6 antibody specificity requires multiple complementary approaches:

• Genetic validation approaches:

  • CRISPR/Cas9 knockout validation: Generate MPHOSPH6-null cells as negative controls

  • siRNA knockdown: Demonstrate reduced signal corresponding to reduced protein expression

  • Overexpression systems: Show increased signal in cells transfected with MPHOSPH6 expression vectors

• Biochemical validation methods:

  • Immunoprecipitation followed by mass spectrometry confirmation

  • Peptide competition assays using the immunizing peptide sequence

  • Western blot correlation with multiple antibodies targeting different epitopes

• Specialized application validation:

  • Cross-application validation: Confirm consistent results across WB, IP, and IF

  • Recombinant protein standards: Use purified MPHOSPH6 as positive controls

  • Orthogonal detection methods: Compare with RNA expression by RT-PCR

• Cross-reactivity assessment:

  • Species cross-reactivity testing in human, mouse, and rat samples

  • Evaluation against related family members to confirm specificity

The validation data should demonstrate consistent detection of the expected 19 kDa band across multiple experimental conditions and cell types .

How do cell cycle phase and phosphorylation status affect MPHOSPH6 antibody detection efficiency?

The detection of MPHOSPH6 by antibodies can be significantly influenced by:

• Cell cycle-dependent phosphorylation modifications:

  • MPHOSPH6 undergoes extensive phosphorylation during M phase

  • This phosphorylation may alter epitope accessibility for certain antibodies

  • Epitopes near phosphorylation sites may show variable detection depending on phosphorylation status

• Methodological considerations for phosphorylation-sensitive detection:

  • Phosphatase treatment controls may help distinguish phosphorylation-dependent epitope masking

  • Synchronized cell populations provide more consistent detection patterns

  • Phase-specific antibodies may be required for certain applications

• Buffer optimization for phosphoprotein preservation:

  • Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate) in all extraction buffers

  • Avoid freeze-thaw cycles that may affect phosphorylation status

  • Consider extracting proteins in denaturing conditions to maintain modifications

• Quantification challenges:

  • Signal intensity may not linearly correlate with protein abundance due to phosphorylation variability

  • Multiple antibodies targeting distinct epitopes can provide more comprehensive detection

  • Normalization against total MPHOSPH6 (using phosphorylation-insensitive antibodies) is recommended

For cell cycle studies, researchers should consider using antibodies raised against sequences distant from known phosphorylation sites for most consistent detection .

What are the implications of MPHOSPH6 gene polymorphisms for antibody epitope recognition and disease association studies?

MPHOSPH6 gene polymorphisms have dual implications for research:

• Impact on antibody epitope recognition:

  • Single nucleotide polymorphisms (SNPs) may alter amino acid sequences within antibody epitopes

  • If polymorphisms occur within the immunogen sequence, antibody binding affinity may be affected

  • Testing antibodies against samples with known polymorphisms can identify recognition limitations

  • Researchers should verify that immunogen sequences (e.g., "MPHOSPH6 fusion protein Ag1080" ) do not overlap with common polymorphic regions

• Disease association implications:

  • Several MPHOSPH6 SNPs have demonstrated significant associations with IgA nephropathy:

    • rs1056654: Associated with a 0.774-fold decrease in IgAN risk (95%CI=0.630-0.952; p=0.015)

    • rs1056675: The "C/C" genotype showed 1.48-fold increased IgAN risk (95%CI=1.03-2.13; p=0.033)

  • Future studies should investigate potential associations with:

    • Cancer susceptibility (preliminary studies in colorectal cancer showed no significant association)

    • Cell cycle dysregulation disorders

    • RNA processing pathologies

• Research design considerations:

  • Population stratification: Different ethnic groups may have different polymorphism frequencies

  • Sample size requirements: Well-powered studies typically require >400 cases and controls

  • Replication studies: Initial findings should be validated in independent populations

These findings highlight the importance of considering genetic variation when selecting antibodies and interpreting experimental results across different populations .

How can MPHOSPH6 antibodies be utilized effectively in multiplex staining and high-throughput screening approaches?

MPHOSPH6 antibodies can be integrated into multiplex and high-throughput workflows through these strategies:

• Multiplex immunofluorescence optimization:

  • Compatible fluorophore selection: MPHOSPH6 antibodies have been successfully conjugated with diverse fluorophores including MaxLight 405, MaxLight 490, MaxLight 550, and MaxLight 650

  • Antibody panel design: Combine with cell cycle markers (cyclin B1, phospho-histone H3) for co-expression analysis

  • Spectral unmixing: Apply computational approaches to separate overlapping fluorescent signals when using multiple markers

• High-throughput assay adaptation:

  • Validated for FluoroSpot assays using different tagging and detection systems:

    • 1×4 configuration (one tagged antigen with four detection reagents)

    • 4×1 configuration (four differently tagged versions with one detection reagent)

    • 4×4 configuration for maximum multiplexing capability

  • Automation compatibility: Works with standard liquid handling systems

  • Miniaturization potential: Successfully used in 384-well plate formats with reduced antibody consumption

• Quantitative analysis approaches:

  • Relative spot volume (RSV) measurements integrate size and fluorescence intensity data

  • Competition between differently tagged versions should be considered during assay design

  • Background control optimization is critical, particularly for the LED380 detection channel

• Cross-platform validation:

  • Results should be confirmed across multiple platforms (e.g., flow cytometry, western blot)

  • Internal standards and controls should be included to normalize between experimental runs

  • Data normalization approaches should account for platform-specific signal characteristics

These multiplex approaches enable simultaneous assessment of MPHOSPH6 with other markers, allowing for more sophisticated understanding of its interactions and regulation .

What are the critical considerations for designing experiments to study MPHOSPH6 expression in clinical samples?

When designing studies of MPHOSPH6 expression in clinical samples, researchers should address:

• Sample collection and preservation protocols:

  • Phosphoprotein preservation: Rapid fixation is essential (≤15 minutes post-excision)

  • Fixative selection: 10% neutral buffered formalin (24-48 hours) is recommended for IHC

  • Snap freezing protocols: Optimal for protein extraction for western blotting

  • Storage conditions: -80°C for frozen samples; paraffin blocks stable at room temperature

• Validated clinical sample types:

  • Human breast cancer tissue has shown consistent positive staining

  • Other tissue types require additional validation

  • Cell type-specific expression patterns should be documented

• Antibody selection for clinical applications:

  • Multiple validated antibodies are available with distinct characteristics:

    • Rabbit polyclonal antibodies: Broader epitope recognition (10695-1-AP)

    • Mouse monoclonals: Higher specificity (sc-377481, sc-81846)

    • Different host species facilitate co-staining with other markers

• Technical validation requirements:

  • Positive and negative control tissues in each experimental run

  • Batch-to-batch consistency assessment

  • Inter-observer scoring standardization for IHC

  • Digital pathology quantification validation

• Clinical correlation considerations:

  • Cell cycle phase standardization between samples

  • Correlation with established cell proliferation markers

  • Integration with clinical patient data

  • Multivariate analysis accounting for confounding variables

For studies linking MPHOSPH6 to disease, findings should be validated across independent patient cohorts of sufficient size to achieve statistical power .

How should researchers approach epitope mapping and functional domain analysis when working with MPHOSPH6 antibodies?

For advanced epitope mapping and functional domain analysis of MPHOSPH6:

• Structural basis for antibody selection:

  • MPHOSPH6 contains several distinct functional domains:

    • RNA-binding region (preferentially binds pyrimidine homopolymers)

    • Nuclear localization signal motif

    • Phosphorylation sites activated during M phase

  • Target antibodies to specific domains based on research questions

• Epitope mapping methodologies:

  • Mutagenesis approaches to identify critical binding residues

  • Competition assays between antibodies recognizing distinct epitopes

  • Hydrogen-deuterium exchange mass spectrometry for detailed epitope characterization

• Functional domain blocking strategies:

  • Antibodies targeting specific domains can serve as functional blockers

  • Domain-specific antibodies can distinguish between different functions:

    • RNA binding activity

    • Protein-protein interaction surfaces

    • Post-translational modification sites

• Cross-species conservation analysis:

  • MPHOSPH6 sequence identity between species:

    • Mouse: 86% identity to human

    • Rat: 87% identity to human

  • Conserved epitopes offer broader species reactivity

  • Species-specific epitopes allow selective targeting

• Technical approach optimization:

  • Recombinant fragment expression for domain-specific antibody generation

  • Epitope tagging strategies for tracking specific domains

  • CRISPR-mediated domain deletion for functional validation

This comprehensive approach enables precise targeting of specific MPHOSPH6 functional domains for mechanistic studies of its diverse cellular roles .