NPHP4 Antibody

What is NPHP4 and why is it significant in research?

NPHP4 encodes a protein involved in renal tubular development and function that interacts with nephrocystin in a multifunctional complex localized to actin- and microtubule-based structures . The significance of NPHP4 in research stems from its association with nephronophthisis type 4 and Senior-Loken syndrome type 4, which presents as a combination of nephronophthisis and retinitis pigmentosa . NPHP4 functions as a crucial component of the selective gate at the transition zone that controls movement of both soluble and membrane-associated proteins between flagellar and cytoplasmic compartments . This barrier function is essential for proper ciliary function, making NPHP4 a focal point in ciliopathy research. The protein is widely distributed in ciliated organisms, and its dysfunction has significant implications for understanding human diseases affecting kidneys and vision .

What applications are NPHP4 antibodies commonly used for?

NPHP4 antibodies are employed across multiple experimental applications. According to available product information, validated applications include:

Immunofluorescence represents the most commonly published application, with at least 10 published studies utilizing NPHP4 antibodies for this purpose . Western blotting applications have been documented in at least one publication . These techniques allow researchers to examine NPHP4 expression patterns, subcellular localization (particularly at the transition zone), and protein-protein interactions in various experimental models.

What are the optimal storage conditions for NPHP4 antibodies?

For maximum stability and performance, NPHP4 antibodies should be stored at -20°C . According to manufacturer specifications, the antibodies remain stable for one year after shipment when stored properly . The storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain antibody integrity during freeze-thaw cycles . For small volume antibodies (20μL), preparations may contain 0.1% BSA as a stabilizer . Importantly, aliquoting is generally unnecessary for -20°C storage of NPHP4 antibodies provided in the glycerol formulation . Researchers should avoid repeated freeze-thaw cycles that could compromise antibody performance and verify specific storage recommendations for their particular antibody product, as formulations may vary slightly between manufacturers.

What positive controls should be used when working with NPHP4 antibodies?

When validating NPHP4 antibodies, researchers should consider using established positive controls. Based on validation data, mouse brain tissue and Y79 cells have been confirmed as positive controls for Western blot applications . For immunohistochemistry, human heart tissue has been successfully used, with optimal results achieved using TE buffer (pH 9.0) for antigen retrieval, although citrate buffer (pH 6.0) may serve as an alternative . For immunofluorescence and immunocytochemistry applications, hTERT-RPE1 cells and MDCK cells provide reliable positive controls . When establishing new experimental systems, researchers should first validate their antibody using these known positive controls before proceeding with experimental samples. Additionally, wild-type samples compared against NPHP4-null samples (when available) serve as excellent specificity controls, as demonstrated in the Chlamydomonas reinhardtii model system where the antibody recognized a band of the expected size in wild-type but not nphp4 whole cells .

How can researchers confirm the subcellular localization of NPHP4?

Determining the precise subcellular localization of NPHP4 requires complementary approaches. Research in Chlamydomonas reinhardtii has established that NPHP4 localizes specifically to the transition zone at the base of cilia/flagella . To confirm this localization pattern, researchers can employ several techniques:

First, immunofluorescence microscopy using both wide-field and specialized methods like total internal reflection fluorescence/epi-fluorescence structured light microscopy (TESM) can provide high-resolution localization data . Co-staining with established transition zone markers such as CEP290 is valuable, as studies have shown NPHP4 localizes distal to CEP290 with a mean peak-to-peak distance of approximately 137 nm . Second, for definitive subcellular localization, pre-embedding immunoelectron microscopy can be performed, which has revealed that NPHP4 is located at the periphery of the distal transition zone, in close association with the transition zone membrane . Third, fractionation studies comparing whole cells, cell bodies, and isolated flagella/cilia can provide biochemical confirmation of localization patterns . In Chlamydomonas, all NPHP4 remained within the cell body fraction after deflagellation, confirming its transition zone rather than ciliary shaft localization .

What methodological approaches can distinguish NPHP4 from other nephrocystins?

Distinguishing NPHP4 from other nephrocystin family members requires careful methodological considerations. First, researchers should select antibodies generated against unique epitopes of NPHP4. For instance, some validated antibodies target the C-terminus of NPHP4 or specific peptide regions like amino acid residues Q1066-I1116 of human NPHP4 (Accession O75161) . Second, molecular weight verification is critical - NPHP4 has a predicted molecular weight of approximately 158 kDa (1426 amino acids) and is typically observed at approximately 150 kDa in Western blots . This size distinction helps differentiate it from other nephrocystins.

Third, co-immunoprecipitation followed by mass spectrometry can confirm protein identity while revealing interaction partners. Fourth, examining localization patterns provides another method for distinction: NPHP4 has a characteristic localization to the transition zone, specifically at the distal part near the membrane, which differs from other nephrocystins . Finally, using genetic models (knockout/knockdown) as negative controls offers definitive validation - antibodies should show no signal in NPHP4-null cells or tissues but maintain normal signals for other nephrocystins . Combined, these approaches provide robust authentication of NPHP4-specific signals.

How does NPHP4 dynamics differ from other transition zone proteins?

NPHP4 demonstrates distinct dynamics compared to other transition zone proteins, particularly CEP290. Research using Chlamydomonas reinhardtii as a model system has revealed fundamental differences in protein stability and turnover . While CEP290 exhibits high dynamism at the transition zone, NPHP4 remains remarkably static . This was demonstrated through elegant zygote experiments where wild-type gametes were mixed with NPHP4-tagged gametes to form quadriflagellated zygotes with a common cytoplasm containing both tagged and untagged NPHP4 .

Even after 60 minutes, only two transition zones showed antibody labeling, indicating minimal incorporation of tagged NPHP4 into previously untagged transition zones . Additionally, the fluorescence intensity remained constant throughout the experiment, suggesting negligible replacement of tagged NPHP4 with untagged protein . This stability contrasts sharply with CEP290's dynamic behavior. Furthermore, localization studies have established that NPHP4 and CEP290 define different domains within the transition zone and assemble independently of each other - NPHP4 localization appears normal in cep290 mutant cells, and CEP290 localization remains unaffected in nphp4 mutant cells . These findings have significant implications for experimental design when studying transition zone assembly and maintenance.

What is the functional significance of NPHP4's role at the transition zone barrier?

Specifically, levels of certain membrane-associated proteins were significantly decreased in nphp4 mutants, while others showed increased abundance . Most notably, these differences were predominantly observed in proteins with molecular weights above 50 kDa, with few differences detected below this threshold . This molecular weight specificity suggests NPHP4 regulates the passage of larger proteins through the transition zone barrier. The functional importance of NPHP4 is further demonstrated by the fact that normal flagellar protein composition was restored in NPHP4-rescued strains . These findings have significant implications for understanding ciliopathies associated with NPHP4 mutations, as protein mislocalization due to defects in the transition zone barrier likely underlies the diverse phenotypic consequences observed in affected patients.

What are common challenges in Western blot detection of NPHP4?

Western blot detection of NPHP4 presents several technical challenges researchers should anticipate. First, NPHP4's large size (approximately 150 kDa observed; 158 kDa calculated) necessitates optimization of gel percentage and transfer conditions . Standard protocols often require modification - using lower percentage gels (6-8%) and extending transfer times with careful temperature control improves detection of such large proteins. Second, due to its membrane association in the transition zone, NPHP4 may require stronger extraction conditions than typical cytosolic proteins . Buffer optimization with adequate detergents is crucial for efficient extraction.

Third, potential cross-reactivity can occur, especially with other nephrocystin family members. Researchers should verify specificity using appropriate controls, ideally including NPHP4-null samples as negative controls . Fourth, degradation products may appear as multiple bands, particularly with suboptimal sample handling. Maintaining samples at appropriate temperatures and including protease inhibitors can minimize this issue. Finally, antibody dilution requires careful optimization - recommended ranges of 1:500-1:1000 for Western blot should be tested and adjusted based on signal-to-noise ratio in each experimental system . Addressing these challenges systematically improves detection reliability and experimental reproducibility.

How can researchers optimize immunofluorescence protocols for NPHP4 detection?

Optimizing immunofluorescence protocols for NPHP4 detection requires attention to several critical parameters. Based on published applications, researchers should consider the following approach:

First, fixation method selection is crucial - paraformaldehyde (typically 4%) preserves NPHP4 epitopes while maintaining cellular architecture, particularly at the transition zone where NPHP4 localizes . Second, permeabilization requires careful optimization; while standard Triton X-100 (0.1-0.5%) works for many applications, gentler detergents may better preserve membrane-associated structures where NPHP4 resides. Third, blocking conditions significantly impact signal-to-noise ratio - extended blocking (1-2 hours) with BSA (3-5%) or normal serum matching the secondary antibody host improves specificity.

Fourth, primary antibody concentration should be titrated within the recommended range (1:10-1:100 for immunofluorescence) , with longer incubation times (overnight at 4°C) often improving signal quality. Fifth, washing steps between antibody applications are critical - multiple extended washes with PBS containing low detergent concentrations reduce background. Finally, researchers should consider co-staining with established markers (e.g., CEP290 for transition zone studies) to provide contextual localization information . When analyzing results, Z-stack imaging through the entire ciliary transition zone region ensures complete visualization of NPHP4's discrete localization pattern.

What methodologies help distinguish specific from non-specific NPHP4 antibody signals?

Distinguishing specific from non-specific NPHP4 antibody signals requires rigorous validation approaches. First, genetic validation using NPHP4-null models represents the gold standard - the absence of signal in knockout/knockdown samples provides definitive evidence of specificity . For instance, in Chlamydomonas studies, antibodies recognized bands of expected size in wild-type but not nphp4 whole cells, with signal restoration in rescued strains .

Second, peptide competition assays, where the antibody is pre-incubated with the immunizing peptide before application, should abolish specific signals while non-specific binding remains. Third, multiple antibody validation involves using different antibodies targeting distinct NPHP4 epitopes - concordant localization patterns strongly support specificity. Fourth, subcellular fractionation confirms whether detected signals correspond to expected localization patterns based on known NPHP4 biology . Finally, signal correlation with protein levels is essential - experiments that modulate NPHP4 expression should show corresponding changes in signal intensity.

Additionally, researchers should evaluate signal patterns against established NPHP4 localization data - specific staining at transition zones (not diffuse cytoplasmic signals) aligns with published findings . Implementing these validation strategies increases confidence in experimental results and supports reproducible research.

How should researchers address antibody cross-reactivity issues with NPHP4?

Addressing antibody cross-reactivity issues with NPHP4 requires systematic evaluation and optimization strategies. First, researchers should conduct comprehensive specificity testing using Western blot analysis across multiple tissues and cell types, comparing observed bands with the expected molecular weight of approximately 150 kDa . Unexpected bands may indicate cross-reactivity with related proteins. Second, bioinformatic analysis of the immunizing peptide sequence against protein databases can identify potential cross-reactive proteins, allowing for more informed interpretation of results.

Third, validation across multiple applications strengthens confidence in specificity - if the same antibody shows consistent and expected patterns in Western blot, immunofluorescence, and immunoprecipitation, specificity is more likely . Fourth, comparing multiple NPHP4 antibodies targeting different epitopes can help distinguish true NPHP4 signals from cross-reactive signals. Fifth, genetic approaches provide definitive validation - testing the antibody in NPHP4-knockdown or knockout models should show corresponding signal reduction or elimination .

Finally, application-specific optimization of protocols, particularly adjusting antibody concentration, incubation times, and washing conditions, can improve signal-to-noise ratio. If cross-reactivity persists despite these measures, researchers should explicitly acknowledge these limitations when reporting results and consider alternative antibodies or complementary non-antibody-based approaches to confirm findings.

How can NPHP4 antibodies be utilized to study protein-protein interactions?

NPHP4 antibodies enable sophisticated studies of protein-protein interactions through several methodological approaches. Co-immunoprecipitation (Co-IP) represents a primary technique, where NPHP4 antibodies can capture NPHP4 along with its binding partners from cell or tissue lysates . This approach has helped establish interactions between NPHP4 and other proteins, including nephrocystin, with which it forms a multifunctional complex .

Proximity ligation assays (PLA) offer another powerful approach, allowing visualization of protein interactions in situ when NPHP4 antibodies are used in conjunction with antibodies against potential interaction partners. This technique is particularly valuable for confirming interactions within specific subcellular regions like the transition zone. Immunofluorescence co-localization studies using NPHP4 antibodies alongside antibodies against other transition zone proteins (such as CEP290) have revealed distinct but related localization patterns, providing insights into functional relationships .

Additionally, NPHP4 antibodies can be utilized in pull-down assays followed by mass spectrometry for unbiased identification of interaction partners. When combined with genetic approaches (such as comparing interactomes in wild-type versus disease-model systems), these methods can reveal how pathogenic mutations affect NPHP4's interaction network. Such studies are critical for understanding the molecular pathways underlying NPHP4-associated diseases.

What insights can NPHP4 antibodies provide about transition zone architecture?

NPHP4 antibodies have been instrumental in elucidating transition zone architecture. High-resolution imaging using these antibodies has established NPHP4 as a marker for the distal part of the transition zone, positioned near the membrane and distal to CEP290 . This precise localization, with a measured mean peak-to-peak distance of approximately 137 nm between CEP290 and the C-terminus of NPHP4, has helped define distinct domains within the transition zone .

Immunoelectron microscopy with NPHP4 antibodies has further refined our understanding, showing that NPHP4 is specifically located at the transition zone periphery, closely associated with the transition zone membrane, which notably remains resistant to nonionic detergent treatment . This peripheral localization aligns with NPHP4's function in regulating protein trafficking across the transition zone barrier.

Studies comparing NPHP4 and CEP290 localization have revealed that these proteins define different transition zone domains and are incorporated independently of each other . The static nature of NPHP4 at the transition zone, contrasting with CEP290's dynamic behavior, suggests different roles in maintaining transition zone architecture . These findings contribute to a more nuanced understanding of transition zone organization and function, with implications for understanding ciliopathies caused by mutations in transition zone components.

How do NPHP4 antibodies contribute to understanding ciliopathies?

NPHP4 antibodies provide critical tools for investigating the molecular mechanisms underlying ciliopathies, particularly nephronophthisis type 4 and Senior-Loken syndrome type 4 . By enabling visualization and biochemical analysis of NPHP4, these antibodies help researchers understand how mutations affect protein localization, stability, and function. Studies using NPHP4 antibodies have established that NPHP4 functions as an essential component of the selective gate at the transition zone, controlling the movement of both soluble and membrane-associated proteins between flagellar and cytoplasmic compartments .

Comparison between wild-type and NPHP4-mutant samples has revealed that loss of NPHP4 function disrupts protein composition in cilia, particularly affecting membrane-associated proteins and allowing abnormal entry of cytosolic proteins larger than 50 kDa into the ciliary compartment . This protein mislocalization likely underlies the diverse phenotypic consequences observed in patients with NPHP4 mutations .

Additionally, NPHP4 antibodies facilitate studies of potential therapeutic interventions by providing readouts for assessing whether treatments restore normal NPHP4 localization and function. The ability to track NPHP4 in patient-derived cells and model systems offers valuable biomarkers for disease progression and therapeutic response, advancing both fundamental understanding of ciliopathies and clinical applications.

What emerging techniques are enhancing NPHP4 antibody applications in research?

Several cutting-edge techniques are expanding the utility of NPHP4 antibodies in research. Super-resolution microscopy methods, including structured illumination microscopy (SIM), stimulated emission depletion (STED), and photoactivated localization microscopy (PALM), are overcoming the diffraction limit to reveal nanoscale details of NPHP4 localization within the transition zone . These approaches build upon earlier work using total internal reflection fluorescence/epi-fluorescence structured light microscopy (TESM) that established precise measurements of NPHP4's position relative to other transition zone components .

Proximity-dependent biotinylation (BioID or TurboID) combined with NPHP4 antibodies for validation is revealing the dynamic protein interaction landscape surrounding NPHP4 at the transition zone. This technique identifies even transient or weak interactions that traditional co-immunoprecipitation might miss. Single-molecule tracking using fluorescently-tagged antibody fragments is providing insights into the remarkably stable nature of NPHP4 at the transition zone, contrasting with the more dynamic behavior of other transition zone proteins like CEP290 .

Additionally, antibody-based proteomics approaches are enabling systematic comparison of ciliary protein composition between normal and NPHP4-deficient samples, expanding upon earlier observations of altered membrane protein content in NPHP4-mutant flagella . Finally, the development of highly specific nanobodies against NPHP4 epitopes promises improved penetration and reduced steric hindrance for studying this important transition zone component in intact cellular contexts.