NPHP4 Antibody, HRP conjugated

What is NPHP4 and why is it a significant research target?

NPHP4 (Nephrocystin-4, also known as nephroretinin) is a 1426 amino acid protein with an observed molecular weight of approximately 150-158 kDa . It plays a critical role in the organization of apical junctions in kidney cells through interactions with NPHP1 and RPGRIP1L/NPHP8 . NPHP4 is essential for normal photoreceptor ribbon synapse maintenance, outer segment formation, and sperm development . Most significantly, NPHP4 is involved in the regulation of mammalian ciliogenesis and ciliary membrane composition as part of a transition zone complex .

Disease-causing variants in the NPHP4 gene are associated with renal ciliopathies, particularly nephronophthisis (NPH), which causes progressive kidney failure . In some cases, NPHP4 mutations can also lead to retinal degeneration without kidney involvement, as observed in canine models . The significance of NPHP4 as a research target stems from its involvement in these pathological conditions and its important role in cellular structures.

What are the standard applications for NPHP4 Antibody, HRP conjugated?

NPHP4 Antibody, HRP conjugated is primarily designed for ELISA applications . The horseradish peroxidase (HRP) conjugation provides direct enzymatic detection capabilities, eliminating the need for secondary antibody incubation steps. While ELISA is the validated application, researchers have also adapted similar antibodies for:

For applications other than ELISA, researchers should conduct preliminary optimization experiments to determine appropriate conditions for the HRP-conjugated format.

What specimens and tissues have shown successful reactivity with NPHP4 antibodies?

Research using NPHP4 antibodies has demonstrated reactivity with several human and animal tissues/cell types:

• Human respiratory epithelial cells - particularly at the transition zone of cilia

• Mouse brain tissue

• Y79 cells (human retinoblastoma cell line)

• Human heart tissue (for IHC applications)

• hTERT-RPE1 cells (human retinal pigmented epithelial cells)

• MDCK cells (canine kidney cells)

In particular, NPHP4 antibodies have been successfully used to study nasal epithelial cells, which provide an accessible sample type for investigating ciliopathies . This approach has clinical significance as it may help secure and accelerate the diagnosis of nephronophthisis by verifying inconclusive genetic results .

How should I design experiments for analyzing NPHP4 expression in patient-derived respiratory epithelial cells?

When designing experiments to analyze NPHP4 expression in respiratory epithelial cells, consider following the methodology validated in recent ciliopathy research :

• Sample Collection and Processing:

  • Collect nasal epithelial cells using a nasal brush or scraping technique

  • Process samples immediately or store appropriately to preserve protein integrity

  • Consider establishing air-liquid interface (ALI) cultures for certain experiments

• Immunofluorescence Protocol:

  • For co-localization studies, use appropriate markers such as acetylated α-tubulin to identify ciliary structures

  • When studying NPHP4 interaction with other proteins (e.g., NPHP1), perform co-staining experiments

  • For deciliation experiments to visualize transition zone components, follow established protocols that allow rupture at the transition zone

• Analysis Approach:

  • Use densitometry to quantify co-localization of NPHP4 with other proteins

  • Compare NPHP4 expression patterns between control and patient samples

  • Consider blinded evaluation to eliminate observer bias

Recent research has demonstrated that NPHP1 and NPHP4 colocalize at the transition zone in respiratory epithelial cells, with overlapping immunofluorescence signals confirmed by densitometry results . This methodological approach can be adapted for experiments using HRP-conjugated antibodies in other detection systems.

What are the optimal conditions for using NPHP4 Antibody, HRP conjugated in ELISA assays?

For optimal ELISA performance with NPHP4 Antibody, HRP conjugated:

• Dilution Optimization:

  • Start with the recommended dilution range of 1:500-1:1000

  • Perform a titration experiment to determine optimal concentration for your specific sample type

  • Consider dot blot analysis prior to full ELISA to determine appropriate dilution

• Buffer Conditions:

  • The antibody is typically provided in 0.01 M PBS, pH 7.4, with 0.03% Proclin-300 and 50% Glycerol

  • For coating and washing steps, standard ELISA protocols apply

  • For blocking, use 1-5% BSA or 5% non-fat dry milk in PBS or TBS to reduce background

• Detection System:

  • Since the antibody is HRP-conjugated, use appropriate substrates like TMB (3,3',5,5'-Tetramethylbenzidine) or ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid))

  • Optimize substrate incubation time based on signal development

  • Consider including positive controls (samples known to contain NPHP4) and negative controls

• Data Analysis:

  • Generate standard curves using recombinant NPHP4 if quantitative analysis is required

  • For comparative studies, normalize data to appropriate housekeeping proteins or total protein content

Remember that optimal dilutions/concentrations should be determined empirically by each laboratory based on specific experimental conditions .

How can I validate the specificity of NPHP4 Antibody, HRP conjugated in my experimental system?

Validating antibody specificity is crucial for ensuring reliable experimental results. For NPHP4 Antibody, HRP conjugated, consider these approaches:

• Positive and Negative Controls:

  • Use samples from individuals with genetically confirmed NPHP4 mutations as negative controls, as they typically show absence of NPHP4 protein

  • Include wild-type samples as positive controls

  • Consider using tissues known to express NPHP4, such as kidney or respiratory epithelial cells

• Knockdown/Knockout Validation:

  • If available, use NPHP4 knockdown or knockout cell lines as specificity controls

  • Compare staining patterns in wild-type versus knockout samples

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess immunizing peptide (if available)

  • Compare signal between peptide-blocked and unblocked antibody

• Cross-Validation with Alternative Antibodies:

  • Compare results with other validated NPHP4 antibodies (non-HRP conjugated)

  • If possible, use antibodies targeting different epitopes of NPHP4

• Western Blot Confirmation:

  • Perform western blotting to confirm the molecular weight of the detected protein (~150-158 kDa)

  • Immunoblotting can be used to verify immunofluorescence results, as demonstrated in recent NPHP4 research

How can NPHP4 Antibody be used to differentiate between various renal ciliopathies?

NPHP4 antibodies can be valuable tools for differentiating between various renal ciliopathies through careful analysis of protein expression patterns:

• Diagnostic Approach:
  Research has demonstrated that individuals with disease-causing variants in NPHP4 show complete absence of NPHP4 protein, while NPHP1 is severely reduced . In contrast, individuals with NPHP1 variants show absence of NPHP1 but variable effects on NPHP4 expression. This differential pattern can help distinguish between these genetic causes.

• Multi-protein Analysis Strategy:
  A comprehensive approach analyzing multiple ciliopathy-related proteins can provide better discrimination between disease subtypes:

  Disease Gene	Effect on NPHP4	Effect on NPHP1	Other Markers
  NPHP4 variants	Complete absence	Severely reduced	Normal ciliary markers
  NPHP1 variants	Variable expression	Complete absence	Normal ciliary markers
  Other ciliopathy genes	Typically normal	Typically normal	Variable effects

• Clinical-Genetic Correlation:
  Importantly, NPHP4 immunostaining of nasal epithelial cells can help secure and accelerate the diagnosis of nephronophthisis—both by verifying inconclusive genetic results and by stratifying genetic diagnostic approaches . This approach has successfully identified two genetically unsolved individuals who were later confirmed to have disease-causing variants in NPHP1 and NPHP4, respectively .

What challenges might arise when using NPHP4 Antibody, HRP conjugated for detection of NPHP4 missense variants?

Detecting NPHP4 missense variants presents unique challenges compared to detecting deletions or truncating mutations:

• Epitope Accessibility Issues:

  • Missense mutations may alter protein folding without eliminating expression

  • These conformational changes might mask the epitope recognized by the antibody

  • Consider using multiple antibodies targeting different regions of NPHP4

• Variable Expression Patterns:

  • Some missense variants may show reduced but not absent protein expression

  • Quantitative analysis becomes more important than simple presence/absence determination

  • Western blotting may provide better quantification than immunofluorescence alone

• Functional Impact Assessment:
  Research has shown that even single missense variants can have significant functional consequences. For example, a homozygous missense variant (c.1027G > A; p.Gly343Arg) in NPHP1 formerly classified as a "variant of unknown significance" showed complete absence of NPHP1 protein in immunofluorescence studies . Similar assessments can be conducted for NPHP4 variants.

• Domain-Specific Effects:
  Consider that missense mutations in different functional domains may have distinct effects:

  • The N-terminal region (first 155 amino acids) of NPHP4 is crucial for interaction with NPHP1

  • Mutations outside this region may preserve NPHP1 interaction but disrupt other functions

  • Mutations in certain domains might affect kidney function, while others might primarily affect retinal function

How should researchers interpret NPHP4 localization data in relation to other transition zone proteins?

Interpreting NPHP4 localization data requires understanding its relationship with other transition zone proteins:

• Spatial Organization:
  NPHP4 localizes distal to the microtubule organizing center (MTOC) of motile cilia, specifically at the transition zone . This localization can be visualized through co-staining with markers such as DNAH5 (an essential component of the MTOC) in deciliated respiratory epithelial cells .

• Protein Interaction Network:

  • NPHP4 and NPHP1 show complete colocalization at the transition zone, with full overlap of immunofluorescence signals

  • NPHP4 also interacts with RPGRIP1, and this interaction is disrupted by mutations in either protein

  • Understanding these interactions helps interpret changes in localization patterns in disease states

• Functional Modules:
  Research provides in vivo evidence for the interaction of NPHP1 and NPHP4 in a functional module . When interpreting localization data, consider that:

  • Changes in one protein may affect localization of interacting partners

  • Different ciliopathy genes may affect distinct functional modules

  • Some proteins may show tissue-specific interaction patterns

• Disease-Specific Patterns:
  The table below summarizes the effect of various disease-causing gene variants on NPHP4 localization:

  Gene Affected	Effect on NPHP4 Localization	Associated Disease
  NPHP4	Absent	NPH, sometimes with retinal involvement
  NPHP1	Present but may show altered pattern	NPH
  BBS genes	Typically normal	Bardet-Biedl Syndrome
  CEP290/NPHP6	Variable effects	JBTS, SLSN

What are the key considerations when using NPHP4 Antibody to study retinal phenotypes in ciliopathies?

When studying retinal phenotypes in ciliopathies using NPHP4 antibodies, researchers should consider:

• Tissue-Specific Expression Patterns:
  NPHP4 is necessary for normal photoreceptor ribbon synapse maintenance and outer segment formation . In retinal tissues, consider:

  • Examining NPHP4 localization in different retinal cell types

  • Comparing expression patterns between affected and unaffected tissues

  • Correlating NPHP4 expression with functional visual assessments

• Genotype-Phenotype Correlations:
  Research in canine models has shown that NPHP4 mutations can cause cone-rod dystrophy without kidney involvement . This suggests:

  • Different mutations in NPHP4 may have tissue-specific effects

  • The N-terminal region (first 155 amino acids) of NPHP4 is crucial for interaction with NPHP1 and kidney phenotypes

  • Mutations outside this region may affect retinal function while preserving renal function

• Model Selection:
  When studying retinal phenotypes, appropriate models include:

  • Human retinal pigmented epithelial cells (hTERT-RPE1)

  • Y79 cells (human retinoblastoma cell line)

  • Canine models with naturally occurring NPHP4 mutations

  • Mouse models with engineered NPHP4 alterations

• Technical Considerations:

  • For immunofluorescence studies in retinal tissues, fixation conditions are critical

  • Consider using both cross-sections and flat mounts for comprehensive analysis

  • Co-staining with photoreceptor markers helps identify cell-specific effects

How can NPHP4 Antibody be applied in nasal epithelial cell analysis for ciliopathy diagnosis?

NPHP4 antibody analysis of nasal epithelial cells represents an innovative approach for ciliopathy diagnosis that is less invasive than kidney biopsy:

• Diagnostic Workflow:
  Recent research has established a diagnostic pathway using nasal epithelial cells :

  • Collect nasal epithelial cells using minimally invasive brushing techniques

  • Perform immunofluorescence analysis with NPHP4 and other ciliopathy-related antibodies

  • Use abnormal staining patterns to guide genetic testing or confirm genetic findings

• Clinical Utility:
  This approach has demonstrated value in:

  • Verifying inconclusive genetic results, such as variants of unknown significance

  • Stratifying genetic diagnostic approaches to prioritize specific genes

  • Accelerating diagnosis in cases with characteristic protein expression patterns

• Protocol Optimization:
  For optimal results:

  • Process samples promptly to preserve protein integrity

  • Use consistent fixation and permeabilization conditions

  • Include appropriate controls (healthy individuals and known mutation carriers)

  • Consider whether HRP-conjugated or standard antibodies are more appropriate for your detection system

• Complementary Approaches:
  Combine antibody analysis with:

  • Ciliary beat pattern analysis via high-speed video microscopy

  • Electron microscopy to examine ultrastructural abnormalities

  • Genetic testing panels targeting known ciliopathy genes

What experimental approaches can investigate the functional relationship between NPHP4 and interacting proteins?

To investigate functional relationships between NPHP4 and its interaction partners:

• Co-immunoprecipitation Studies:

  • Use NPHP4 antibodies to pull down protein complexes

  • Identify binding partners through mass spectrometry

  • Verify interactions through reciprocal co-immunoprecipitation

  • Map interaction domains using truncated protein constructs

• Proximity Labeling Approaches:

  • Employ BioID or APEX2 proximity labeling systems fused to NPHP4

  • Identify proteins in close proximity to NPHP4 in living cells

  • Compare proximity interactomes in different cell types and conditions

• Live Cell Imaging:

  • Create fluorescently tagged NPHP4 and interacting proteins

  • Monitor co-localization and dynamics during ciliogenesis

  • Use FRAP (Fluorescence Recovery After Photobleaching) to assess mobility and binding kinetics

• Functional Rescue Experiments:
  Research has shown that NPHP1 and NPHP4 interact in a functional module . To investigate:

  • Express wild-type NPHP4 in cells from patients with NPHP4 mutations

  • Assess whether this rescues NPHP1 localization

  • Test whether overexpression of interacting partners can compensate for NPHP4 deficiency

• Domain Mapping:
  Studies have refined the NPHP1-binding region of NPHP4 to the first 155 amino acids . Similar approaches can:

  • Generate deletion constructs of NPHP4

  • Test their ability to bind interaction partners

  • Map functional domains responsible for different cellular processes

How should researchers address discrepancies between NPHP4 antibody results and genetic findings?

When faced with discrepancies between NPHP4 antibody results and genetic findings:

• Review Genetic Testing Completeness:

  • Standard genetic testing may miss deep intronic variants, promoter mutations, or structural variants

  • Consider whether whole genome sequencing might detect variants missed by targeted approaches

  • Evaluate whether copy number variation analysis was performed

• Assess Technical Factors:

  • Review antibody specificity and sensitivity in your experimental system

  • Consider whether the epitope recognized by the antibody might be preserved in some mutations

  • Evaluate sample quality and handling procedures

• Consider Alternative Mechanisms:
  Recent research identified two genetically unsolved individuals with aberrant NPHP4 immunostaining who were later found to have disease-causing variants in NPHP1 and NPHP4, respectively . This suggests:

  • Protein-level analysis can sometimes detect abnormalities missed by initial genetic testing

  • Post-translational regulation may affect protein expression independent of coding mutations

  • Variants in regulatory regions may affect expression without changing the coding sequence

• Follow-up Strategy:
  When discrepancies occur:

  • Perform additional protein analyses using antibodies targeting different epitopes

  • Consider expanded genetic testing focused on regions suggested by protein findings

  • Evaluate related proteins in the same pathway or complex

What factors affect the sensitivity and specificity of NPHP4 detection in different experimental systems?

Multiple factors influence the performance of NPHP4 antibodies across experimental systems:

• Antibody Characteristics:

  • The immunogen used to generate the antibody determines epitope specificity

  • Polyclonal antibodies (like the HRP-conjugated NPHP4 antibody) recognize multiple epitopes, increasing sensitivity but potentially reducing specificity

  • Antibody affinity affects detection threshold and signal-to-noise ratio

• Sample Preparation:

  • Fixation methods can mask epitopes or create artifacts

  • For NPHP4 detection in tissues, antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0 may be necessary

  • Permeabilization conditions affect antibody access to intracellular epitopes

• Expression Level Variability:

  • NPHP4 expression levels vary between tissues and cell types

  • Expression may be developmentally regulated or affected by cellular conditions

  • Detection sensitivity must be calibrated to the expected expression level

• Detection System Considerations:
  For HRP-conjugated antibodies:

  • Substrate selection affects sensitivity and dynamic range

  • Signal amplification methods can enhance detection of low-abundance proteins

  • High concentrations of HRP can lead to excessive background or rapid substrate depletion

• Cross-Reactivity Assessment:

  • Validate specificity across species if working with non-human samples

  • Test for potential cross-reactivity with related proteins in the nephrocystin family

  • Include appropriate negative controls to identify non-specific signals

How can researchers optimize protocols when working with challenging sample types for NPHP4 detection?

When working with challenging samples for NPHP4 detection:

• Limited Sample Material (e.g., patient biopsies):

  • Consider multiplex staining to maximize information from minimal tissue

  • Use signal amplification systems like tyramide signal amplification

  • Optimize antibody concentration through careful titration experiments

  • Consider automated staining platforms for consistency with precious samples

• Highly Ciliated Tissues:
  For respiratory epithelial cells or other ciliated tissues:

  • Optimize fixation to preserve ciliary structures while maintaining epitope accessibility

  • Consider deciliation techniques to visualize the transition zone

  • Evaluate both intact tissues and dissociated cells for comprehensive analysis

• Tissues with High Autofluorescence:

  • When using non-HRP conjugated antibodies for fluorescence microscopy:

    • Include appropriate autofluorescence reduction steps (Sudan Black B treatment)

    • Consider spectral unmixing during image acquisition

    • Use fluorophores with emission spectra distinct from tissue autofluorescence

• Formalin-Fixed, Paraffin-Embedded (FFPE) Tissues:

  • Optimize antigen retrieval conditions (pH, temperature, duration)

  • Consider longer primary antibody incubation times (overnight at 4°C)

  • Test signal amplification systems to overcome reduced epitope availability

• Protocol Modifications for Specific Applications:
  For challenging detection scenarios with HRP-conjugated antibodies:

  • In high-background tissues, increase blocking time and concentration

  • For weak signals, extend substrate development time or use more sensitive substrates

  • In tissues with endogenous peroxidase activity, include appropriate quenching steps