IQCB1 Antibody

What is IQCB1 and why is it significant for research?

IQCB1, also known as NPHP5 (nephrocystin-5), is a protein encoded by the IQCB1 gene located on human chromosome 3q21.3. This protein contains IQ calmodulin-binding motifs and plays crucial roles in primary cilia structure and function. IQCB1 is particularly significant for research because mutations in this gene cause Senior-Løken syndrome, a ciliopathy characterized by kidney filtration defects and retinal degeneration. Recent research has also identified IQCB1 as a potential diagnostic and prognostic marker in liver cancer .

What applications are IQCB1 antibodies validated for?

IQCB1 antibodies have been validated for multiple research applications including:

• Western blotting (WB): Typically at dilutions of 1:1000-1:5000

• Immunohistochemistry (IHC): For both frozen and paraffin-embedded tissues

• Immunocytochemistry/Immunofluorescence (ICC/IF): For cellular localization studies

• Enzyme-linked immunosorbent assay (ELISA): For quantitative protein detection

Most commercially available IQCB1 antibodies show reactivity with human samples, though some may cross-react with mouse and rat IQCB1 due to sequence homology .

What are the optimal fixation conditions for IQCB1 immunostaining?

For optimal IQCB1 detection in immunostaining applications:

• For cultured cells: 4% paraformaldehyde for 10-15 minutes at room temperature preserves ciliary and centrosomal structures

• For tissue sections: 4% paraformaldehyde followed by gentle permeabilization is recommended

• Avoid harsh fixatives like methanol for extended periods, as they can disrupt the native conformation of ciliary proteins

• For double immunostaining with centrosomal or ciliary markers, sequential antibody incubation may yield better results than simultaneous application .

How can I optimize IQCB1 antibody detection in ciliary structures?

Optimizing detection of IQCB1 in ciliary structures requires specific methodological considerations:

• Sample preparation: Use confluent, serum-starved cells (24-48h) to promote ciliogenesis before fixation

• Antigen retrieval: For paraffin sections, citrate buffer (pH 6.0) heat-induced epitope retrieval improves signal

• Signal amplification: For low expression samples, consider using biotin-streptavidin amplification or tyramide signal amplification

• Co-localization studies: Use established ciliary markers like ARL13B alongside IQCB1 antibodies

• Microscopy settings: Employ confocal microscopy with z-stack acquisition to properly visualize the three-dimensional ciliary structures

Research has demonstrated that IQCB1 primarily localizes to the ciliary base and transition zone, with some protein also detected in the cytoplasm .

What are the best strategies for validating IQCB1 antibody specificity?

Multiple validation approaches should be employed to ensure IQCB1 antibody specificity:

For instance, in IQCB1 knockdown experiments using shRNA, researchers observed reduced IQCB1 immunostaining at the ciliary base, confirming antibody specificity .

How can IQCB1 antibodies be used to study protein-protein interactions in ciliary research?

IQCB1 antibodies can be effectively employed to study protein interactions using these approaches:

• Co-immunoprecipitation (Co-IP): IQCB1 antibodies can pull down interaction partners. Studies have successfully used this approach to identify EPB41L5 as an IQCB1-interacting protein .

• Proximity ligation assay (PLA): This technique can visualize interactions between IQCB1 and putative binding partners in situ with high sensitivity.

• Immunofluorescence co-localization: Using dual labeling with IQCB1 antibodies and antibodies against potential interactors, researchers have shown IQCB1 co-localization with CEP290 at the ciliary base .

• Domain mapping: By combining IQCB1 antibodies with expression of truncated constructs, researchers identified that:

  • The N-terminal FERM domain of EPB41L5 interacts with IQCB1

  • The internal domain of IQCB1 containing three IQ motifs and a coiled-coil domain (amino acids 287–443) mediates this interaction

How are IQCB1 antibodies used in cancer research?

IQCB1 antibodies serve as valuable tools in cancer research, particularly for liver hepatocellular carcinoma (LIHC):

What methodologies use IQCB1 antibodies to study ciliopathies?

IQCB1 antibodies are instrumental in studying ciliopathies through various approaches:

• Patient-derived cell models: Researchers have used IQCB1 antibodies to characterize ciliary abnormalities in cells derived from patients with NPHP5-LCA (IQCB1-associated Leber congenital amaurosis) .

• Rescue experiments: IQCB1 antibodies help validate gene augmentation approaches by confirming protein expression and proper localization after genetic intervention .

• Tissue-specific phenotyping: Immunohistochemistry with IQCB1 antibodies in retinal and renal tissues helps characterize tissue-specific manifestations of ciliopathies.

• Developmental studies: IQCB1 antibody staining during development helps track temporal expression patterns relevant to ciliopathy pathogenesis .

• Animal models: Immunostaining with cross-reactive IQCB1 antibodies in animal models of ciliopathies helps validate disease mechanisms and test therapeutic approaches .

How can IQCB1 antibodies be used to develop prognostic models for liver cancer?

Research demonstrates that IQCB1 antibodies contribute to prognostic model development for liver cancer:

How can I overcome detection sensitivity issues with IQCB1 antibodies?

Detection sensitivity challenges with IQCB1 antibodies can be addressed through several methodological approaches:

• Endogenous detection limitations: Some researchers have noted that "immunostaining of endogenous IQCB1 using a commercial anti-IQCB1 antibody was below the detection limit" . To overcome this:

  • Use overexpression systems with tagged IQCB1 for localization studies

  • Employ signal amplification methods like tyramide signal amplification

  • Consider using more sensitive detection methods such as proximity ligation assay

• Optimizing antibody concentration: Titration experiments are essential, with recommended dilutions varying by application:

  • For Western blot: 1:1000-1:5000

  • For immunofluorescence: 1:10-1:100 (potentially requiring higher concentrations than other applications)

• Sample preparation optimization:

  • For cultured cells: Serum starvation to enhance cilia formation

  • For tissues: Careful fixation and optimized antigen retrieval

What are the best controls for IQCB1 antibody experiments?

Implementing appropriate controls is critical for reliable IQCB1 antibody experiments:

• Positive controls:

  • Cell lines with confirmed IQCB1 expression: HEK-293 and HeLa cells show detectable IQCB1 expression in Western blot

  • Tissue positive controls: Human retina shows strong cytoplasmic positivity in cones

• Negative controls:

  • Primary antibody omission control

  • Isotype control (matched rabbit IgG)

  • IQCB1 knockdown/knockout cells or tissues

• Expression vector controls:

  • For interaction studies, empty vector controls should be included alongside IQCB1 expression constructs

  • When studying domain interactions, truncated constructs should be verified for expression level and stability

• Co-localization controls:

  • Known ciliary markers (ARL13B, acetylated tubulin)

  • Centrosomal markers (γ-tubulin, NEDD1) for basal body localization

How do I interpret conflicting IQCB1 antibody staining patterns?

When faced with conflicting IQCB1 antibody staining patterns, consider these analytical approaches:

• Epitope mapping: Different antibodies may recognize distinct epitopes that could be differentially accessible depending on:

  • Protein conformation

  • Interaction with binding partners

  • Post-translational modifications

• Subcellular localization variability: IQCB1 localizes to multiple compartments:

  • Primarily at the ciliary base/transition zone

  • In cytoplasmic puncta

  • At the centrosome

  • In the nucleoplasm, microtubules, and cytokinetic bridge in some cell types

• Expression level considerations:

  • Overexpression may alter normal localization patterns

  • Low endogenous expression might require more sensitive detection methods

• Cell type specificity: IQCB1 expression and localization patterns vary across cell types:

  • Renal epithelial cells show primary ciliary localization

  • Photoreceptor cells show connecting cilium localization

  • U-2 OS cells show localization to nucleoplasm, microtubules, cytokinetic bridge, and mitotic spindle

How can IQCB1 antibodies be used to study immune infiltration in cancer?

Recent research reveals novel applications for IQCB1 antibodies in studying immune infiltration in cancer:

• Correlation with immune cell populations: IQCB1 expression has been correlated with immune cell infiltration in liver cancer. Specifically, a positive correlation was observed between IQCB1 expression and T-helper 2 (Th2) cells, while a negative correlation was found with Th17 cells .

• Checkpoint mechanisms: IQCB1 antibodies can help investigate associations between IQCB1 expression and immune checkpoint molecules. Studies have identified a positive association between IQCB1 and immune checkpoints, particularly with CD276 .

• Tumor microenvironment analysis: Single-cell data analysis combined with IQCB1 antibody staining has revealed widespread expression of IQCB1 in the tumor microenvironment .

• Therapeutic relevance: IQCB1 antibody-based screening has identified potential therapeutic compounds. Research found 12 drugs related to IQCB1, with 10 showing negative correlations and 2 showing positive correlations .

What are the methodological considerations for studying IQCB1 interactions with other ciliary proteins?

Studying IQCB1 interactions with other ciliary proteins requires specific methodological considerations:

• Domain mapping strategies: Research has identified important interaction domains:

  • The N-terminal FERM domain of EPB41L5 (amino acids 1–239) interacts with IQCB1

  • The internal domain of IQCB1 containing three IQ motifs and a coiled-coil domain (amino acids 287–443) mediates interaction with EPB41L5

• Functional interaction studies: IQCB1 antibodies have revealed that:

  • EPB41L5 suppresses IQCB1 localization at the ciliary base

  • Expression of the N-terminal FERM-FA domain of EPB41L5 was sufficient to reduce IQCB1 accumulation at the ciliary base

  • EPB41L5 binding to the IQ-coiled-coil domain suppresses centrosomal association of IQCB1

• Co-immunoprecipitation protocols: Optimized protocols for IQCB1 interactions include:

  • Transient expression in HEK293 cells

  • Lysis in buffers containing 0.5% Triton X-100

  • Use of anti-tag antibodies (anti-FLAG or anti-myc) for immunoprecipitation

  • Analysis of co-precipitation after 24 hours of expression

How can IQCB1 antibodies contribute to gene therapy validation for ciliopathies?

IQCB1 antibodies serve as critical tools for validating gene therapy approaches for IQCB1-associated ciliopathies:

• Expression verification: Following gene augmentation therapy, IQCB1 antibodies confirm successful protein expression in target tissues.

• Localization assessment: Proper subcellular localization of the therapeutic protein to ciliary structures can be verified using immunofluorescence with IQCB1 antibodies.

• Functional rescue evaluation: IQCB1 antibodies help assess whether gene therapy restores proper protein interactions and ciliary function through co-localization and co-immunoprecipitation studies.

• Long-term expression monitoring: Sequential sampling and IQCB1 antibody detection can track the persistence of therapeutic protein expression over time.

• Model validation: Studies have used in vitro disease modeling with patient-derived cells to characterize cilia abnormalities in human NPHP5-LCA, validating gene augmentation as a prospective treatment approach .