NPHP4 Antibody, FITC conjugated

What is NPHP4 and why is it significant in ciliopathy research?

NPHP4 (Nephronophthisis 4) is a protein that plays a crucial role in the function of ciliary transition zones. It is part of a heterogeneous group of inherited renal ciliopathies characterized by progressive kidney failure. Research has shown that NPHP4 functions as a crucial component of the selective gate at the transition zone that controls the movement of both soluble and membrane-associated proteins between flagellar and cytoplasmic compartments . Mutations in the NPHP4 gene cause nephronophthisis and visual impairment in humans, making it a significant target for studying the molecular basis of these conditions . NPHP4 co-localizes with other transition zone proteins like NPHP1, and this interaction has been demonstrated through immunofluorescence studies of respiratory epithelial cells .

What are the primary applications for FITC-conjugated NPHP4 antibodies?

FITC-conjugated NPHP4 antibodies are primarily utilized in direct immunofluorescence applications where the inherent fluorescence properties eliminate the need for secondary antibodies. The applications include:

• Immunofluorescence microscopy of ciliated cells to study NPHP4 localization at the transition zone

• Co-localization studies with other ciliary proteins

• ELISA assays for protein detection

• Flow cytometry analysis of cells expressing NPHP4

The antibody specifically recognizes amino acids 397-543 of human NPHP4 and has been validated for human samples . Studies have shown this antibody can effectively visualize NPHP4 at the base of cilia and flagella, particularly in the transition zone region, which appears as a distinct punctate pattern approximately 137 nm distal to CEP290 localization .

What sample types work best with NPHP4 antibody immunofluorescence?

For optimal results with NPHP4 antibodies in immunofluorescence applications, researchers should consider:

• Respiratory epithelial cells: Nasal epithelial cells have proven highly effective for detecting NPHP4 localization, as demonstrated in studies of nephronophthisis diagnosis .

• Kidney cell lines: Given NPHP4's role in renal function, kidney cell lines such as MDCK cells have shown good antigen recognition.

• Ciliated model organisms: Chlamydomonas reinhardtii has been successfully used to study NPHP4 localization at the transition zone of flagella .

Sample preparation is critical. Respiratory epithelial cells cultured in air-liquid interface (ALI) systems preserve ciliary structures and allow for clear visualization of the transition zone where NPHP4 localizes . For deciliation studies, controlled rupture of the transition zone retains NPHP4 at the apical membrane, enabling detailed analysis of its spatial relationships with other proteins .

How should researchers optimize fixation protocols for NPHP4 antibody staining?

Optimization of fixation protocols is essential for successful NPHP4 immunostaining:

When studying the transition zone specifically, researchers should consider that NPHP4 localizes distal to the microtubule organizing center. This has been confirmed through co-staining with markers like acetylated α-tubulin and CEP290 . Proper permeabilization with 0.1-0.2% Triton X-100 after fixation ensures antibody access to the transition zone region without disrupting structures.

What are the recommended dilutions and controls for FITC-conjugated NPHP4 antibody?

While optimal working dilutions should be determined experimentally for each application, typical starting dilutions include:

• Immunofluorescence: 1:50 to 1:200

• ELISA: 1:500 to 1:2000

Essential controls include:

• Negative controls:

  • Secondary antibody only (for unconjugated primary antibodies)

  • Isotype control (rabbit IgG-FITC)

  • Samples from NPHP4 knockout or mutant organisms

• Positive controls:

  • Wild-type ciliated respiratory epithelial cells

  • Cell lines with confirmed NPHP4 expression

  • NPHP4-HAC or NPHP4-HAN tagged rescued strains

Studies have validated antibody specificity using nphp4 mutant cells, which show absence of NPHP4 signal in immunofluorescence, while wild-type cells display distinct localization at the transition zone . This control approach confirms antibody specificity and provides confidence in experimental findings.

How can researchers effectively design co-localization studies with NPHP4 and other ciliary proteins?

Designing effective co-localization studies requires careful selection of compatible antibodies and appropriate imaging protocols:

• Selecting compatible primary antibodies:

  • When using FITC-conjugated NPHP4 antibody, pair with primary antibodies raised in species other than rabbit

  • Consider using antibodies against known transition zone proteins like NPHP1, CEP290, or RPGRIP1L

  • For studies of ciliary structure, incorporate antibodies against acetylated α-tubulin (ciliary axoneme) or DNAH5 (microtubule organizing center)

• Image acquisition parameters:

  • Use sequential scanning to prevent bleed-through between fluorophores

  • Employ high-resolution techniques such as structured illumination microscopy or TESM (total internal reflection fluorescence/epi-fluorescence structured light microscopy) to resolve the narrow transition zone region

  • Collect z-stacks with 0.2-0.3 μm steps to fully capture the three-dimensional organization

• Quantitative analysis:

  • Measure peak-to-peak distances between NPHP4 and other transition zone proteins

  • Apply densitometry to confirm co-localization, as demonstrated in studies showing NPHP1 and NPHP4 overlap

  • Use Pearson's correlation coefficient or Manders' overlap coefficient to quantify the degree of co-localization

Research has shown that NPHP1 and NPHP4 demonstrate complete signal overlap at the transition zone, with densitometry confirming their co-localization . In contrast, CEP290 localizes approximately 137 nm proximal to NPHP4, allowing for detailed mapping of transition zone architecture .

What approaches can distinguish between dynamic and static components of the transition zone using NPHP4 antibodies?

Understanding the dynamics of transition zone proteins provides insights into ciliary assembly and maintenance. Research using NPHP4 antibodies has revealed distinct dynamics compared to other transition zone proteins:

• Zygote formation experimental design:

  • Mix gametes with differentially tagged proteins (e.g., wild-type and NPHP4-HA tagged)

  • Form quadriflagellated zygotes where two transition zones contain wild-type NPHP4 and two contain tagged NPHP4

  • Track protein exchange between transition zones over time (20, 40, 60 minutes)

  • Compare dynamics with other transition zone proteins like CEP290

• FRAP (Fluorescence Recovery After Photobleaching):

  • Photobleach FITC-conjugated NPHP4 antibody signal at specific transition zones

  • Monitor fluorescence recovery over time

  • Calculate recovery half-time and mobile fraction

• Analysis of protein dynamics:

  • Measure signal intensity at specific timepoints

  • Compare with known dynamic proteins (e.g., CEP290) and static proteins

Studies using these approaches have demonstrated that NPHP4 remains static at the transition zone, showing little to no exchange between wild-type and tagged transition zones even after 60 minutes, in stark contrast to the highly dynamic behavior of CEP290 . This static nature suggests NPHP4 may serve as a structural component of the transition zone gate.

How can NPHP4 antibodies be used to investigate protein transport defects in ciliopathies?

NPHP4 antibodies can reveal critical insights into protein transport defects associated with ciliopathies:

• Comparative proteomic analysis:

  • Isolate flagella/cilia from wild-type and NPHP4 mutant samples

  • Separate proteins into detergent-soluble membrane-plus-matrix and axonemal fractions

  • Compare protein profiles using SDS-PAGE and identify differences

  • Perform mass spectrometry to identify specific proteins affected by NPHP4 deficiency

• Protein classification by localization defects:

  • Identify proteins with decreased abundance in NPHP4 mutant flagella (particularly membrane-associated proteins)

  • Identify cytosolic proteins abnormally present in mutant flagella (typically >50kDa)

  • Use immunofluorescence to validate localization changes

• Rescue experiments:

  • Reintroduce wild-type NPHP4 into mutant cells

  • Verify restoration of normal ciliary protein composition

  • Analyze which transport defects are reversible

Research comparing wild-type and nphp4 mutant flagella has revealed that NPHP4 loss causes significant changes in the membrane-plus-matrix fraction, with some proteins showing decreased levels (particularly membrane-associated proteins) while others (typically large cytosolic proteins above 50kDa) abnormally accumulate in flagella . This provides strong evidence that NPHP4 functions as a critical component of the ciliary gate that selectively controls protein passage.

How can NPHP4 immunofluorescence be applied to accelerate the diagnosis of nephronophthisis?

NPHP4 immunofluorescence of respiratory epithelial cells offers a valuable diagnostic approach:

• Sample collection and preparation:

  • Obtain nasal epithelial cells via minimally invasive brushing

  • Establish air-liquid interface cultures or process for immediate analysis

  • Perform immunofluorescence staining with NPHP4 and NPHP1 antibodies

• Diagnostic criteria:

  • Complete absence of NPHP4 staining in individuals with NPHP4 disease-causing variants

  • Severely reduced NPHP1 staining in individuals with NPHP4 genotype

  • Absence of NPHP1 in individuals with NPHP1 disease-causing variants

• Complementary diagnostic approaches:

  • Combine immunofluorescence results with western blotting validation

  • Use findings to guide genetic testing for specific NPH genes

  • Incorporate clinical features and family history

Studies examining 86 individuals with genetically determined renal ciliopathies have demonstrated that immunofluorescence analysis of NPHP4 and NPHP1 in respiratory epithelial cells provides a reliable diagnostic approach . This method has successfully identified individuals with disease-causing variants, including those with variants previously classified as "variants of unknown significance," thereby accelerating diagnosis and genetic counseling .

What experimental approaches can verify interactions between NPHP4 and other transition zone proteins?

Verifying protein-protein interactions involving NPHP4 requires multiple complementary approaches:

• Co-immunoprecipitation strategies:

  • Use anti-NPHP4 antibodies to pull down protein complexes

  • Perform western blotting for suspected interaction partners (e.g., NPHP1)

  • Include appropriate controls (IgG control, lysates from NPHP4 mutant cells)

• Proximity labeling techniques:

  • Generate NPHP4 fusion proteins with BioID or APEX2

  • Identify proteins in close proximity through biotinylation

  • Validate candidates using co-localization studies with FITC-conjugated NPHP4 antibodies

• Genetic interaction studies:

  • Compare phenotypes of single mutants (nphp4, nphp1, cep290) versus double mutants

  • Examine protein localization patterns in different genetic backgrounds

  • Test if proteins localize independently or interdependently

Research has demonstrated that NPHP4 and NPHP1 form a functional module at the transition zone, with NPHP4 depletion causing severe reduction of NPHP1, while NPHP4 and CEP290 localize to the transition zone independently of each other . These findings highlight the complexity of transition zone architecture and protein interactions, with important implications for understanding the molecular basis of ciliopathies.

What are common technical issues with NPHP4 immunofluorescence and how can they be resolved?

Researchers may encounter several challenges when performing NPHP4 immunofluorescence:

For optimal results with FITC-conjugated antibodies specifically, researchers should protect samples from light during all steps and consider photobleaching effects during imaging. Using freshly prepared samples and storing antibodies according to manufacturer recommendations (typically with 50% glycerol at -20°C) helps maintain signal quality .

How can researchers distinguish between specific and non-specific binding when using NPHP4 antibodies?

Distinguishing specific from non-specific binding requires rigorous validation:

• Genetic controls:

  • Compare staining patterns between wild-type and NPHP4 knockout/mutant samples

  • Verify complete absence of signal in mutants with verified null mutations

  • Use rescued mutants (NPHP4-R) to confirm specificity

• Peptide competition assays:

  • Pre-incubate antibody with excess immunizing peptide (AA 397-543)

  • Perform parallel staining with blocked and unblocked antibody

  • Specific signal should be eliminated by peptide competition

• Multiple antibody validation:

  • Compare staining patterns using antibodies targeting different NPHP4 epitopes

  • Verify consistent localization patterns across antibodies

  • Cross-validate with tagged NPHP4 constructs (e.g., NPHP4-HAC, NPHP4-HAN)

Studies using nphp4 mutant cells have conclusively demonstrated antibody specificity, as staining is completely absent in these cells but present at the transition zone in wild-type samples . Additionally, complementation with tagged NPHP4 constructs restores the expected localization pattern, further confirming specificity .

How can NPHP4 antibodies contribute to understanding ciliopathy disease mechanisms?

NPHP4 antibodies enable deeper understanding of ciliopathy mechanisms through several advanced approaches:

• Investigation of variant pathogenicity:

  • Compare NPHP4 localization between wild-type and cells expressing variants of unknown significance

  • Assess interactions with partner proteins like NPHP1

  • Correlate protein mislocalization with clinical phenotypes

• Ciliary gate functional studies:

  • Analyze protein composition of cilia/flagella in different genetic backgrounds

  • Categorize proteins by their dependence on NPHP4 for proper localization

  • Map the NPHP4-dependent "gatekeeper" function at the molecular level

• Disease modeling:

  • Generate patient-derived respiratory epithelial cells

  • Apply NPHP4 immunofluorescence to validate disease models

  • Test therapeutic approaches by monitoring NPHP4/NPHP1 localization restoration

Research has demonstrated that NPHP4 variants affect not only NPHP4 localization but also that of interaction partners like NPHP1, providing in vivo evidence for their functional relationship . This approach has successfully verified the pathogenicity of variants previously classified as uncertain, highlighting the value of protein localization studies in variant interpretation .

What experimental approaches can investigate the differential dynamics of transition zone proteins using NPHP4 antibodies?

Understanding the contrasting dynamics of transition zone proteins provides insights into ciliary assembly and maintenance:

• Comparative dynamics studies:

  • Design pulse-chase experiments with inducible tagged proteins

  • Compare incorporation rates of newly synthesized NPHP4 versus other transition zone proteins

  • Analyze protein turnover during ciliary maintenance and regeneration

• Multi-protein tracking during ciliogenesis:

  • Use FITC-conjugated NPHP4 antibody alongside differently labeled antibodies against CEP290, NPHP1, and other transition zone components

  • Track protein recruitment during ciliogenesis

  • Establish temporal assembly sequence

• Stability analysis after perturbation:

  • Apply treatments that disrupt ciliary structure (deciliation)

  • Monitor NPHP4 retention at basal bodies compared to other proteins

  • Analyze recovery patterns during reciliation

Research has revealed striking differences between transition zone proteins, with NPHP4 remaining static at the transition zone while CEP290 demonstrates high dynamism . These differential dynamics suggest distinct roles: CEP290 may participate in signaling or regulatory functions requiring rapid exchange, while NPHP4 likely provides structural stability to the ciliary gate .

How can super-resolution microscopy enhance NPHP4 transition zone research?

Super-resolution microscopy techniques offer unprecedented insights into transition zone architecture:

• STORM/PALM applications:

  • Achieve 20-30 nm resolution of transition zone proteins

  • Map precise three-dimensional organization of NPHP4 relative to other components

  • Reveal subdomains within the transition zone

• Expansion microscopy approach:

  • Physically expand samples to increase effective resolution

  • Maintain NPHP4 antibody compatibility through gentle fixation

  • Achieve detailed visualization of transition zone protein networks

• Live-cell super-resolution techniques:

  • Track dynamic interactions between NPHP4 and mobile components

  • Monitor structural changes during ciliary assembly

  • Visualize protein transport through the transition zone

TESM (total internal reflection fluorescence/epi-fluorescence structured light microscopy) has already revealed that NPHP4 localizes approximately 137 nm distal to CEP290 . Future application of even higher resolution techniques promises to provide more detailed protein maps of the transition zone and improve understanding of how mutations disrupt this organization in disease states.

What approaches combine NPHP4 immunofluorescence with functional ciliary studies?

Integrating structural and functional analyses provides comprehensive insights:

• Ciliary protein trafficking assays:

  • Track fluorescently labeled cargo proteins in wild-type versus NPHP4 mutant backgrounds

  • Quantify transport rates and accumulation patterns

  • Correlate trafficking defects with structural abnormalities visualized by NPHP4 immunofluorescence

• Intraflagellar transport (IFT) analysis:

  • Combine NPHP4 immunofluorescence with live imaging of IFT components

  • Investigate how transition zone integrity affects IFT train assembly and movement

  • Analyze if specific IFT components are differentially affected by NPHP4 depletion

• Cilium-dependent signaling pathway assessment:

  • Examine Hedgehog, Wnt, and PDGF pathway activity in cells with NPHP4 deficiency

  • Correlate transition zone structural defects with signaling abnormalities

  • Test whether restoring NPHP4 localization rescues signaling defects