MPRIP Antibody

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

MPRIP (Myosin Phosphatase Rho-Interacting Protein) is a multifunctional protein that has gained research significance due to its dual localization in both cytoplasmic and nuclear compartments. Initially characterized as a cytoplasmic protein localizing to F-actin stress fibers, recent research has revealed its presence in nuclear structures where it undergoes liquid-liquid phase separation (LLPS) . This protein interacts with PIP2 (phosphatidylinositol 4,5-bisphosphate), RNA polymerase II (RNAPII), and the nuclear myosin I isoform MYO1C, suggesting a critical role in transcriptional regulation . The discovery of MPRIP's nuclear functions has opened new avenues for investigating nuclear architecture and gene expression control mechanisms.

What epitopes are targeted by commercially available MPRIP antibodies?

Commercial MPRIP antibodies target various regions of the protein, offering researchers flexibility in experimental design. For example, the rabbit polyclonal antibody ABIN2787432 targets the middle region of MPRIP with the amino acid sequence "ATISAIEAMK NAHREEMERE LEKSQRSQIS SINSDIEALR RQYLEELQSV" . Other available antibodies target the N-terminal region, C-terminal region, or specific amino acid sequences such as AA 473-486, AA 771-820, and AA 723-772 . Some antibodies also recognize phosphorylated epitopes, including pSer232 and pThr231 . This diversity allows researchers to select antibodies that best match their experimental requirements, particularly when investigating domain-specific functions or post-translational modifications.

What species reactivity should be considered when selecting an MPRIP antibody?

Species reactivity is a critical consideration when selecting an MPRIP antibody for cross-species research. The ABIN2787432 antibody demonstrates broad cross-reactivity with human (100%), cow (93%), pig (93%), horse (93%), mouse (86%), rat (86%), guinea pig (86%), rabbit (86%), and dog (86%) MPRIP proteins . This reactivity profile is based on sequence homology analysis of the targeted epitope across species. When planning experiments involving multiple model organisms, researchers should verify the conservation of the epitope sequence to ensure consistent detection. For specialized applications requiring species-specific detection, antibodies with narrower reactivity profiles might be preferable to minimize cross-reactivity issues.

How should MPRIP antibodies be validated before use in critical experiments?

Proper validation of MPRIP antibodies before use in critical experiments requires a multi-step approach:

• Western blot validation: Confirm specificity by detecting a single band at the expected molecular weight (~120 kDa for full-length MPRIP) . Include positive controls (tissues/cells known to express MPRIP) and negative controls (knockout or knockdown samples if available).

• Immunofluorescence validation: Verify subcellular localization patterns by comparing with published literature. MPRIP should display both cytoplasmic stress fiber localization and granular nuclear patterns .

• Blocking peptide competition: Preincubate the antibody with its immunizing peptide to confirm signal specificity.

• Testing on fractionated samples: Analyze nuclear and cytoplasmic fractions separately, using GAPDH (cytoplasmic) and Lamin B (nuclear) as fraction purity controls .

• Cross-validation: Compare results using multiple antibodies targeting different MPRIP epitopes.

This comprehensive validation strategy ensures reliable data interpretation in subsequent experiments.

What are the optimal conditions for using MPRIP antibodies in immunofluorescence?

For optimal immunofluorescence results with MPRIP antibodies, researchers should follow this validated protocol:

• Sample preparation: Culture cells (e.g., U2OS) on high-performance cover glasses with restricted thickness-related tolerance (depth = 0.17 mm ± 0.005 mm) and specific refractive index (1.5255 ± 0.0015) to ensure optimal imaging quality .

• Fixation and permeabilization: Fix cells with 4% formaldehyde for 20 minutes, followed by permeabilization with 0.1% Triton X-100 for 5 minutes .

• Blocking: Block non-specific binding using 5% Bovine Serum Albumin (BSA) in PBS .

• Antibody incubation: Apply anti-MPRIP antibody (e.g., HPA022901) at an optimized dilution (typically 1:100 to 1:500) .

• Washing: Perform five 5-minute washes in PBS with 0.1% Tween between each step for super-resolution microscopy applications .

• Mounting: Mount samples in 90% glycerol with 4% n-Propyl gallate (NPG) to enhance signal preservation and reduce photobleaching .

This protocol enables detection of both cytoplasmic and nuclear MPRIP populations, with nuclear MPRIP displaying a characteristic granular pattern dispersed in the nucleoplasm.

What is the recommended protocol for MPRIP detection by Western blotting?

For optimal Western blot detection of MPRIP, follow this methodological approach:

• Sample preparation: For total cellular MPRIP, use standard whole-cell lysates. For compartment-specific analysis, prepare subcellular fractions, ensuring fraction purity by probing for compartment-specific markers (e.g., Lamin B for nuclear fraction, GAPDH for cytoplasmic fraction) .

• Sample loading: Load 20-50 μg protein per lane. MPRIP has a molecular weight of approximately 120 kDa .

• Electrophoresis conditions: Use 7.5-10% SDS-PAGE gels to achieve optimal resolution of high molecular weight MPRIP.

• Transfer parameters: Transfer to PVDF membrane at 100V for 2 hours using cold transfer buffer containing 20% methanol.

• Blocking conditions: Block with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Antibody incubation: Dilute primary MPRIP antibody (e.g., HPA022901) to manufacturer-recommended concentration (typically 1:1000) in 5% BSA-TBST and incubate overnight at 4°C.

• Detection system: Use HRP-conjugated secondary antibodies and enhanced chemiluminescence for visualization.

The affinity-purified nature of commercial MPRIP antibodies generally results in clean detection with minimal background .

How can MPRIP antibodies be utilized to study liquid-liquid phase separation in the nucleus?

MPRIP undergoes liquid-liquid phase separation (LLPS) in the nucleus, forming dynamic condensates with liquid-like properties. To investigate this phenomenon:

• Live cell imaging approach: Transfect cells with GFP-tagged MPRIP (pDEST53-GFP-MPRIP) and perform time-lapse confocal microscopy, capturing images every 5 minutes for 8+ hours . This allows visualization of the formation, fusion, and disassembly of MPRIP condensates.

• LLPS verification: Treat cells with 5% 1,6-hexanediol, which disrupts weak hydrophobic interactions that drive phase separation. Monitor rapid dissolution of MPRIP condensates to confirm their LLPS nature .

• Mobility analysis: Perform Fluorescence Recovery After Photobleaching (FRAP) experiments on MPRIP condensates to measure protein mobility and exchange rates. MPRIP condensates show time-dependent solidification, with increasing immobile fractions over time .

• Domain contribution: Express truncated versions of MPRIP (N-terminal 1-450 and C-terminal 450-1000) to determine which regions drive phase separation. The intrinsically disordered regions (IDRs) predicted by bioinformatics analysis are likely responsible for LLPS behavior .

• Co-condensation studies: Use dual-color imaging with markers of known nuclear condensates to identify potential co-phase separation with other nuclear factors.

This multi-faceted approach provides mechanistic insights into MPRIP's role in forming dynamic nuclear compartments.

What methodological approaches can be used to study MPRIP's interaction with nuclear actin?

To investigate MPRIP's interaction with nuclear actin, researchers can employ these methodological approaches:

• Co-immunoprecipitation (Co-IP): Prepare nuclear lysates from cell lines (e.g., HeLa), incubate with anti-MPRIP antibody (2 μg) overnight at 4°C, then with G-protein magnetic beads for 1 hour. After washing, analyze the precipitated complexes by Western blotting for actin .

• Proximity ligation assay (PLA): Use MPRIP antibody in combination with actin antibody to visualize and quantify direct interactions in situ, providing spatial information about interaction sites within the nucleus.

• F-actin cosedimentation assay: Incubate purified MPRIP protein with preassembled F-actin, then ultracentrifuge to separate bound and unbound fractions. Analyze the pellet and supernatant for MPRIP to determine binding affinity.

• Live-cell imaging with dual labeling: Express fluorescently tagged MPRIP and nuclear actin markers to track their dynamic relationship. The formation of MPRIP fibers and their relationship to nuclear actin can be monitored by time-lapse microscopy, as MPRIP shows unique behavior of binding to fibrous structures while maintaining liquid-like properties .

• Domain mapping: Express truncated versions of MPRIP to identify specific domains responsible for actin binding, focusing on regions with predicted actin-binding motifs.

These approaches provide complementary information about the molecular details and functional consequences of MPRIP-actin interactions in the nucleus.

How can researchers investigate MPRIP's role in RNAPII-mediated transcription?

To investigate MPRIP's involvement in RNAPII-mediated transcription, researchers can implement these methodological approaches:

• Co-immunoprecipitation of transcriptional complexes: Immunoprecipitate MPRIP from nuclear extracts and probe for RNAPII (especially the active form phosphorylated at Ser5) and MYO1C/NM1. This approach has confirmed that MPRIP forms complexes with both proteins .

• Chromatin immunoprecipitation (ChIP): Perform ChIP using anti-MPRIP antibodies to identify genomic regions associated with MPRIP. Compare these with RNAPII-bound regions to establish correlation.

• Nascent RNA analysis: Combine MPRIP knockdown/knockout with techniques that measure nascent transcription (e.g., EU-seq, NET-seq) to quantify the impact of MPRIP depletion on transcription rates.

• Super-resolution microscopy: Apply STED microscopy to visualize the spatial relationship between MPRIP, PIP2-rich nuclear lipid islets (NLIs), and active RNAPII. Statistical analysis using Manders and Spearman coefficients can quantify colocalization .

• Functional studies: Employ MPRIP depletion or overexpression followed by RNA-seq to identify genes whose expression depends on MPRIP function.

• Domain-specific mutations: Introduce mutations in MPRIP's PH domain to disrupt PIP2 binding and evaluate the impact on RNAPII association and transcriptional activity.

This integrated approach can establish the mechanistic role of MPRIP in connecting nuclear actin, PIP2, and transcriptional machinery.

What are common pitfalls in MPRIP immunofluorescence experiments and how can they be addressed?

When performing MPRIP immunofluorescence, researchers may encounter these challenges:

• High cytoplasmic background obscuring nuclear signal:

  • Problem: MPRIP's abundant cytoplasmic localization can mask its nuclear presence.

  • Solution: Optimize permeabilization conditions (try 0.5% Triton X-100 for nuclear proteins), use confocal or super-resolution microscopy, and analyze optical sections through the nucleus . Consider nuclear-cytoplasmic fractionation before fixation.

• Variable detection of nuclear MPRIP:

  • Problem: Nuclear MPRIP may be detected inconsistently between experiments.

  • Solution: Ensure antibodies recognize the nuclear-localized epitope (verify NLS region accessibility) . Use multiple antibodies targeting different regions. Standardize fixation protocols, as overfixation may mask epitopes.

• Cross-reactivity with other PH domain-containing proteins:

  • Problem: Antibodies may detect other proteins with similar domains.

  • Solution: Validate specificity using MPRIP knockdown/knockout controls. Perform peptide competition assays to confirm signal specificity.

• Difficulty detecting phase-separated condensates:

  • Problem: MPRIP condensates may be disrupted during fixation.

  • Solution: Use live-cell imaging with GFP-MPRIP . If fixation is necessary, use methods that preserve phase-separated structures (e.g., pre-extraction protocols or specialized fixatives).

• Loss of signal during washing steps:

  • Problem: Excessive washing may remove weakly bound antibodies.

  • Solution: Optimize washing conditions - use five 5-minute washes in PBS with 0.1% Tween between steps . Consider increasing primary antibody concentration or incubation time.

These strategies will improve detection reliability and data interpretation in MPRIP immunofluorescence experiments.

How should researchers interpret heterogeneous MPRIP distribution patterns in different cell types?

When interpreting heterogeneous MPRIP distribution patterns across cell types, consider these methodological approaches:

• Quantitative analysis framework:

  • Establish a standardized classification system for MPRIP distribution patterns (e.g., predominantly cytoplasmic, predominantly nuclear, equal distribution, condensate-forming).

  • Quantify relative fluorescence intensity between compartments using image analysis software.

  • Calculate nuclear/cytoplasmic ratios across multiple cells (n>50) to establish statistically valid distribution profiles.

• Cell cycle-dependent variation:

  • Co-stain for cell cycle markers to determine if MPRIP localization correlates with specific cell cycle phases.

  • Synchronize cells and analyze MPRIP distribution at defined time points after release.

• Differentiation status correlation:

  • For primary cells or differentiation models, correlate MPRIP distribution patterns with differentiation markers.

  • Analyze whether changes in MPRIP localization precede or follow cell fate transitions.

• Isoform-specific distribution:

  • Different MPRIP isoforms may show distinct localization patterns. Use isoform-specific antibodies or expression constructs to determine isoform-specific distribution .

  • Consider that MPRIP's human isoform 3 has been specifically identified in nuclear condensates .

• Post-translational modification analysis:

  • Investigate whether phosphorylation status correlates with localization patterns using phospho-specific antibodies .

  • Treat cells with kinase or phosphatase inhibitors to determine if phosphorylation regulates MPRIP localization.

This systematic approach allows for meaningful interpretation of cell type-specific MPRIP distribution patterns in relation to cellular physiology and function.

What control experiments are essential when studying the specificity of MPRIP antibodies in co-immunoprecipitation studies?

When performing co-immunoprecipitation (Co-IP) experiments with MPRIP antibodies, these essential controls ensure data reliability:

• Input sample control:

  • Always run an input sample (5-10% of the lysate used for IP) to confirm the presence of MPRIP and potential interacting partners before immunoprecipitation.

  • This establishes the baseline abundance of proteins and aids in calculating IP efficiency.

• Negative antibody control:

  • Perform parallel IP with isotype-matched IgG from the same species as the MPRIP antibody.

  • This controls for non-specific binding to antibody or beads and is essential for distinguishing true interactions.

• MPRIP knockdown/knockout control:

  • When available, include lysates from MPRIP-depleted cells as a negative control.

  • This verifies antibody specificity and confirms that detected interactions depend on MPRIP presence.

• Reciprocal co-IP:

  • Validate key interactions by performing the reverse immunoprecipitation (e.g., IP with anti-RNAPII or anti-MYO1C antibodies and blot for MPRIP).

  • Confirmation of interaction in both directions strongly supports true association.

• Competitive peptide blocking:

  • Pre-incubate MPRIP antibody with excess immunizing peptide before IP.

  • This should abolish specific binding and confirm epitope specificity.

• Stringency controls:

  • Perform parallel IPs with increasing salt concentrations (150 mM, 300 mM, 450 mM NaCl).

  • This distinguishes direct, high-affinity interactions from weak or indirect associations.

When interpreting co-IP data for MPRIP interactions with RNAPII and MYO1C, as described in the literature , these controls ensure that the observed associations represent genuine biological interactions rather than technical artifacts.