MAPKBP1 Antibody

What is MAPKBP1 and what are its key structural domains?

MAPKBP1 (Mitogen-Activated Protein Kinase Binding Protein 1) is a 170 kDa scaffolding protein consisting of three main domains: an N-terminal WD40 domain, a central c-Jun N-terminal kinase (JNK)-binding domain, and a C-terminal coiled-coil dimerization domain . The protein exists in multiple alternatively spliced variants with calculated molecular weights ranging from 109 kDa to 164 kDa, though the observed molecular weight in experimental conditions is typically 200-230 kDa . MAPKBP1 functions as a scaffolding protein for JNK signaling pathways and has recently been characterized as a microtubule-binding protein with cell cycle-dependent localization to centrosomes, basal bodies, and mitotic spindle poles .

What types of MAPKBP1 antibodies are currently available for research?

Several types of MAPKBP1 antibodies are available for research applications:

Most commonly available antibodies are rabbit polyclonal antibodies recognizing various epitopes, including N-terminal regions, specific amino acid sequences (e.g., AA 1-100, AA 1-1015), or full-length protein . These antibodies have been validated for applications including Western Blotting, Immunohistochemistry, ELISA, and Immunofluorescence studies .

What are the optimal working dilutions for different MAPKBP1 antibody applications?

Recommended working dilutions vary by application and specific antibody:

Researchers should note that optimal working dilutions should be determined empirically for each experimental system . For IHC applications specifically, antigen retrieval methods may influence antibody performance, with some protocols recommending TE buffer pH 9.0 or citrate buffer pH 6.0 .

How should MAPKBP1 antibodies be stored to maintain optimal activity?

MAPKBP1 antibodies are typically supplied in buffered aqueous glycerol solutions (often PBS with 0.02% sodium azide and 50% glycerol at pH 7.3) . The recommended storage temperature is -20°C, where they remain stable for approximately one year after shipment . Aliquoting is generally unnecessary for -20°C storage, though for antibodies provided at high concentrations, creating working aliquots may be beneficial to prevent freeze-thaw cycles that could compromise activity .

How can researchers validate MAPKBP1 antibody specificity in their experimental systems?

Validating MAPKBP1 antibody specificity requires a multi-faceted approach:

• Western blot verification: Compare observed molecular weight (200-230 kDa) with predicted sizes (109-164 kDa) .

• Positive and negative controls: Use tissues known to express MAPKBP1 (e.g., liver tissue in mice and rats has shown positive results) .

• Immunostaining patterns: Validate subcellular localization patterns. Wild-type MAPKBP1 should show:

  • Centrosomal localization in non-dividing cells

  • Microtubule association in a filamentous pattern

  • Mitotic spindle pole localization during cell division

• Mutant protein controls: If available, cells expressing truncated MAPKBP1 proteins should show altered localization patterns with increased cytosolic/nuclear distribution and reduced centrosomal accumulation .

• Orthogonal validation: Consider RNA interference or CRISPR-based approaches to knock down MAPKBP1 and confirm reduction in antibody signal .

What cellular localization patterns should researchers expect when using MAPKBP1 antibodies in immunofluorescence studies?

MAPKBP1 exhibits distinct localization patterns that vary with cell cycle stage:

• In non-dividing cells:

  • Centrosomal localization (colocalization with pericentrin)

  • Three-dotted pattern or ring structure decorating mother centrioles

  • Smaller accumulations at daughter centrioles

  • Filamentous pattern emerging from centrosomal regions

• In ciliated cells:

  • Basal body localization

  • Microtubule association

• During mitosis:

  • Strong accumulation at mitotic spindle poles from prophase to anaphase

  • Increased cytoplasmic staining

Researchers should note that wild-type and mutant MAPKBP1 show markedly different localization patterns. C-terminally truncated variants (as found in patients with nephronophthisis) display predominantly cytosolic and nuclear localization with occasional midbody accumulation in dividing cells .

How can MAPKBP1 antibodies be used to study nephronophthisis and renal pathology?

MAPKBP1 antibodies are valuable tools for investigating nephronophthisis (NPH), particularly the late-onset, cilia-independent form associated with MAPKBP1 mutations:

• Mutation analysis correlation: Researchers can use MAPKBP1 antibodies to assess protein expression in patient samples with known genetic variants. Truncating mutations often result in either absence of detectable protein or expression of shortened variants .

• Centrosomal recruitment studies: Since proper centrosomal localization of MAPKBP1 is critical for its function, antibodies can help evaluate the impact of disease-associated mutations on protein localization .

• Microtubule association: MAPKBP1 antibodies can reveal defects in microtubule binding capacity of mutant proteins, which may contribute to disease pathogenesis .

• Cell cycle regulation analysis: Since MAPKBP1 shows cell cycle-dependent localization patterns, antibodies can help examine potential mitotic defects in cells harboring MAPKBP1 mutations .

• Domain-specific functions: Using antibodies targeting different epitopes, researchers can investigate the functions of various MAPKBP1 domains and their contributions to disease when mutated .

What signaling pathways associated with MAPKBP1 can be investigated using these antibodies?

MAPKBP1 has been implicated in several signaling pathways that can be investigated using appropriate antibodies:

These pathways were identified in gene expression studies comparing high versus low MAPKBP1 expression in CN-AML patients . Researchers can use MAPKBP1 antibodies in combination with antibodies against key components of these pathways to elucidate the molecular mechanisms underlying MAPKBP1's functions in both normal and pathological conditions.

How can researchers distinguish between different MAPKBP1 isoforms in their experiments?

Differentiating between MAPKBP1 isoforms requires strategic antibody selection and analytical techniques:

• Isoform-specific antibodies: Select antibodies targeting regions that differ between isoforms. For instance, some commercial antibodies specifically target N-terminal regions (N-Term) or defined amino acid sequences (AA 1-100, AA 1-1015) .

• Western blot analysis: Utilize high-resolution SDS-PAGE (6-8% gels) to separate high molecular weight isoforms. MAPKBP1 isoforms can range from 109 kDa to 164 kDa (calculated), though observed molecular weights are typically 200-230 kDa .

• Two-dimensional gel electrophoresis: Combine isoelectric focusing with SDS-PAGE to separate isoforms based on both charge and molecular weight differences.

• Immunoprecipitation followed by mass spectrometry: For definitive isoform identification, immunoprecipitate MAPKBP1 using validated antibodies and perform mass spectrometric analysis to identify specific isoform sequence variations.

• RT-PCR validation: Complement protein-level studies with transcript analysis using isoform-specific primers to confirm the presence of specific splice variants in your experimental system.

What methodological approaches can resolve contradictory findings when studying MAPKBP1 cellular localization?

Researchers may encounter contradictory findings regarding MAPKBP1 localization, as demonstrated by differences between earlier studies reporting no centrosomal localization and later work confirming such localization . To resolve such contradictions:

• Expression level considerations: Endogenous MAPKBP1 expression may be too low for reliable detection in some systems. Compare antibody staining with overexpression of fluorescently-tagged constructs, noting that "very low expression of endogenous MAPKBP1 under nonstressed conditions" might explain some discrepancies .

• Cell cycle-dependent analysis: MAPKBP1 localization varies with cell cycle stage. Synchronize cells and examine localization at specific cell cycle phases using markers like PCNT (for centrosomes) and appropriate cell cycle indicators .

• Fixation method optimization: Test multiple fixation protocols, as some centrosomal proteins are sensitive to specific fixatives. Compare paraformaldehyde, methanol, and glutaraldehyde fixation results.

• Super-resolution microscopy: Utilize techniques like structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy to resolve fine subcellular structures that might be missed by conventional microscopy.

• Live cell imaging: Employ live cell imaging of fluorescently-tagged MAPKBP1 to track dynamic localization changes that might be missed in fixed cell preparations.

• Multiple antibody validation: Use antibodies targeting different epitopes of MAPKBP1 to confirm localization patterns and rule out epitope-specific artifacts .

How can MAPKBP1 antibodies be utilized in studies of protein-protein interactions and complex formation?

MAPKBP1 forms homodimers and interacts with paralogous proteins like WDR62. Researchers can leverage antibodies to study these interactions through:

• Co-immunoprecipitation (Co-IP): Use MAPKBP1 antibodies to pull down protein complexes, followed by Western blotting for interaction partners. This approach has been used to demonstrate MAPKBP1 homodimerization and heterodimerization with WDR62 .

• Proximity ligation assay (PLA): This technique can visualize protein-protein interactions in situ with high sensitivity. Use antibodies against MAPKBP1 and potential interaction partners, followed by oligonucleotide-linked secondary antibodies that generate a fluorescent signal when proteins are in close proximity.

• FRET/BRET analysis: For live cell studies, combine antibody-based validation with fluorescence or bioluminescence resonance energy transfer methods using tagged proteins to monitor interactions in real-time.

• Domain-specific studies: Compare wild-type MAPKBP1 with truncated variants lacking specific domains to determine their contribution to protein interactions. For example, truncation of the C-terminal coiled-coil domain abrogates dimerization ability .

• Centrosome isolation: Combined with antibody-based detection methods, centrosome isolation protocols can help identify MAPKBP1 interactions specifically within this organelle.

What considerations should researchers take into account when designing experiments to study MAPKBP1 in kidney disease models?

When investigating MAPKBP1 in kidney disease contexts, particularly nephronophthisis:

• Age-appropriate models: MAPKBP1-associated nephronophthisis typically presents as juvenile or late-onset disease, unlike other forms of NPH. Models should reflect this temporal aspect, with analysis extending into adulthood .

• Cell type considerations: Focus on appropriate kidney cell types, particularly those involved in NPH pathogenesis. Inner medullary collecting duct (IMCD) cells have been successfully used in MAPKBP1 studies .

• Patient-derived resources: Where possible, use patient-derived cells (fibroblasts, induced pluripotent stem cells, or kidney organoids) with known MAPKBP1 mutations .

• Domain-specific analysis: Design experiments that investigate all three major domains of MAPKBP1, as "all protein domains are indispensable for appropriate MAPKBP1 intracellular localization and function" .

• Transcriptomic integration: Combine protein-level studies with RNA-seq analysis, as has been done using primary patient fibroblasts to elucidate "consequences of aberrant intracellular trafficking" .

• Cilia-independent mechanisms: Unlike most forms of NPH, MAPKBP1-associated disease is considered "cilia-independent." Experimental designs should consider both ciliary and non-ciliary functions of MAPKBP1, including its roles in cell cycle regulation, microtubule organization, and JNK signaling .