KIAA0319 Antibody

What is KIAA0319 and why is it significant in neurodevelopmental research?

KIAA0319 is a transmembrane protein associated with dyslexia susceptibility, containing several polycystic kidney disease (PKD) domains that may mediate interactions between neurons and glial fibers during development . It plays critical roles in neuronal migration and cortical development, making it an important target for neurodevelopmental research. The protein exists in multiple variants, with the full-length form (variant A) being 1052 amino acids (115.8 kDa), while variants B and C are 958 amino acids (104.9 kDa) and 991 amino acids (108.4 kDa), respectively . Understanding KIAA0319's function has implications for elucidating the biological basis of reading disabilities and related cognitive disorders.

What types of KIAA0319 antibodies are currently available for research?

Several KIAA0319 antibodies are available for research applications, varying in host species, clonality, and epitope recognition:

How do I select the appropriate KIAA0319 antibody for my specific research needs?

Selection of the appropriate KIAA0319 antibody should be based on several factors:

• Species compatibility: Ensure the antibody recognizes KIAA0319 in your experimental model. For example, NB100-93472 is validated for mouse and predicted to work with rat models , while CAC11321 targets human KIAA0319 .

• Application requirements: Consider which techniques you'll be using. Some antibodies perform better in certain applications, such as NB100-93472 which is validated for IHC, IHC-P, ELISA, and WB .

• Epitope recognition: Different antibodies target different regions of KIAA0319. If studying specific variants or domains, select an antibody that recognizes your region of interest. For instance, antibodies targeting the N-terminal region can detect extracellular epitopes in non-permeabilized conditions .

• Validation data: Review available validation data. For example, NB100-93472 detects a ~150 kDa band in mouse brain lysates by Western blot , which aligns with the expected molecular weight of KIAA0319.

What are the recommended protocols for Western blot detection of KIAA0319?

For optimal Western blot detection of KIAA0319:

• Sample preparation: Use RIPA buffer for protein extraction from brain tissue or neural cells. For brain samples, 35μg of total protein is typically sufficient .

• Antibody concentration: For NB100-93472, use 0.5-1.5 μg/ml for Western blot applications . Optimization may be required for other antibodies.

• Gel conditions: Use 6-8% SDS-PAGE gels to properly resolve the large KIAA0319 protein (~115-150 kDa).

• Reducing vs. non-reducing conditions: Consider running samples under both reducing and non-reducing conditions, as KIAA0319 forms dimers that can be visualized under non-reducing conditions. Under reducing conditions, expect to see four distinct bands for the full-length protein, while under non-reducing conditions, the higher molecular weight bands (dimers) become more prominent .

• Detection method: Chemiluminescence provides good sensitivity for detecting KIAA0319 .

• Expected results: A band at approximately 150 kDa for full-length KIAA0319 in brain tissue, with possible additional bands (an unknown identity band at ~25 kDa has been consistently observed) .

How should I optimize immunostaining protocols for KIAA0319 detection?

For successful immunostaining of KIAA0319:

• Fixation: Use 4% paraformaldehyde for 15-20 minutes at room temperature to preserve protein structure while maintaining epitope accessibility.

• Antibody dilution: Start with recommended dilutions (e.g., 1:10-1:500 for IHC applications with NB100-93472) and optimize as needed.

• Permeabilization considerations:

  • For detecting total KIAA0319 expression, use standard permeabilization with 0.1-0.2% Triton X-100.

  • To specifically detect cell surface KIAA0319, perform staining without permeabilization, which will only detect the extracellular N-terminal region .

• Blocking: Use 5% BSA or normal serum (from species different than the host of the primary antibody) to reduce background.

• Controls: Include appropriate negative controls (secondary antibody only) and positive controls (tissues known to express KIAA0319, such as brain sections).

• Antigen retrieval for paraffin sections: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is recommended for optimal staining in paraffin-embedded tissues .

How can I validate the specificity of KIAA0319 antibodies in my experimental system?

To confirm antibody specificity:

• Peptide competition assays: Pre-incubate the antibody with its immunizing peptide (if available) to block specific binding. This has been successfully demonstrated for antibodies like NB100-93472, where blocking with the immunizing peptide eliminates the specific 150 kDa band .

• Multiple antibody validation: Use different antibodies targeting distinct epitopes of KIAA0319 and compare staining patterns. If consistent patterns are observed, this increases confidence in specificity. For example, similar patterns were observed with antisera against different regions of KIAA0319 (R1, R2) and with tag-specific antibodies in transfected cells .

• Knockout/knockdown controls: If available, use KIAA0319 knockout or knockdown samples as negative controls.

• Recombinant expression: Express tagged versions of KIAA0319 in cell lines and verify co-localization of antibody staining with tag-specific antibodies. This approach confirmed that both N-terminal antibodies and C-terminal tag antibodies detected the same protein in transfected cells .

• Cross-reactivity testing: Test the antibody on samples from multiple species to confirm predicted cross-reactivity. For example, NB100-93472 was validated in mouse and predicted to work in rat due to 100% sequence homology .

How can I distinguish between different KIAA0319 isoforms using antibodies?

To distinguish between KIAA0319 isoforms:

• Epitope-specific antibodies: Select antibodies targeting regions that differ between isoforms. For instance, antibodies targeting the transmembrane domain would recognize full-length KIAA0319 (variant A) but not variants B and C, which lack this domain .

• Molecular weight discrimination: Full-length KIAA0319 (KA) is approximately 115.8 kDa, while variants B and C are 104.9 kDa and 108.4 kDa respectively (without signal peptide) . Western blot can differentiate these based on molecular weight.

• Subcellular localization: Different isoforms localize to different cellular compartments. The full-length KA localizes to the plasma membrane, while KB and KC (lacking the transmembrane domain) are retained in the endoplasmic reticulum . Using immunofluorescence under permeabilized vs. non-permeabilized conditions can help distinguish these patterns.

• Non-permeabilized staining: Only plasma membrane-localized isoforms with extracellular domains (like the full-length KA) will be detected in non-permeabilized immunofluorescence, allowing discrimination from ER-retained isoforms .

What strategies can I employ to study KIAA0319 endocytosis and trafficking?

To investigate KIAA0319 endocytosis and trafficking:

• Antibody internalization assays: Incubate live cells with KIAA0319 antibodies at 4°C (where endocytosis is inhibited), then warm to 37°C to allow internalization to resume. The antibody will be internalized along with KIAA0319, allowing visualization of endocytic vesicles .

• Fab fragment controls: To ensure that antibody binding itself doesn't artificially trigger endocytosis, use Fab fragments derived from affinity-purified antibodies as controls .

• Co-localization studies: Perform double immunofluorescence with markers for different cellular compartments (e.g., endosomes, Golgi, ER) to track KIAA0319 trafficking. The full-length KIAA0319 has been shown to co-localize with ER and trans-Golgi network markers in addition to plasma membrane localization .

• Rab5 co-expression: Co-express KIAA0319 with GFP-tagged constitutively active (Q79L) or dominant negative (S34N) Rab5 mutants to investigate endosomal trafficking dynamics, as Rab5 regulates early endosome fusion .

• Live cell imaging: Use fluorescently tagged KIAA0319 constructs for real-time visualization of trafficking and endocytosis in live cells.

How can I investigate the dimerization and post-translational modifications of KIAA0319?

To study KIAA0319 dimerization and modifications:

• Reducing vs. non-reducing SDS-PAGE: Run protein samples under both reducing and non-reducing conditions. Under non-reducing conditions, KIAA0319 dimers remain intact and appear as higher molecular weight bands (>270 kDa) .

• Co-immunoprecipitation: Co-express differently tagged versions of KIAA0319 (e.g., myc+His and EGFP tags) and perform co-immunoprecipitation to confirm protein-protein interactions. Pull down with one tag and detect with the other to verify dimerization .

• Glycosylation analysis: KIAA0319 is heavily glycosylated. Treat samples with glycosidases (PNGase F for N-linked glycans, O-glycosidase for O-linked glycans) prior to Western blot to assess the contribution of glycosylation to apparent molecular weight.

• Site-directed mutagenesis: Introduce mutations at potential glycosylation or dimerization sites to assess their impact on protein function and localization. For example, the Y995A mutation has been used to study KIAA0319 functional domains .

• Cross-linking assays: Use chemical cross-linkers to stabilize protein-protein interactions before cell lysis to capture transient or weak interactions between KIAA0319 molecules.

Why do I observe multiple bands when using KIAA0319 antibodies in Western blot?

Multiple bands in KIAA0319 Western blots can occur due to several factors:

• Protein isoforms: KIAA0319 exists in multiple splice variants (A, B, C) with different molecular weights .

• Dimerization: KIAA0319 forms dimers that appear as higher molecular weight bands, especially under non-reducing conditions. For the full-length protein (KA), four bands are typically observed under reducing conditions, with the two larger bands being approximately twice the size of the smaller ones, suggesting dimeric forms .

• Post-translational modifications: KIAA0319 is heavily glycosylated, which can create heterogeneity in apparent molecular weight.

• Proteolytic processing: KIAA0319 may undergo proteolytic cleavage during sample preparation or naturally within cells.

• Non-specific binding: Some antibodies may recognize related proteins. For example, NB100-93472 consistently detects an additional 25 kDa band of unknown identity in mouse brain lysates .

When investigating these multiple bands, consider running appropriate controls, using different antibodies targeting different epitopes, and employing treatments like deglycosylation to help identify the nature of each band.

What are common pitfalls in KIAA0319 antibody-based experiments and how can I avoid them?

Common pitfalls and their solutions include:

• Poor signal-to-noise ratio:

  • Solution: Optimize blocking conditions (5% BSA or normal serum), antibody concentration, and incubation times. For NB100-93472, use the recommended concentration of 0.5-1.5 μg/ml for Western blot .

• False negative results in membrane protein detection:

  • Solution: Ensure proper sample preparation to avoid protein aggregation. For KIAA0319, avoid boiling samples before loading as this can cause transmembrane proteins to aggregate.

• Inconsistent results between experiments:

  • Solution: Standardize protocols, use consistent antibody lots, and include positive controls (e.g., mouse brain lysate for NB100-93472) .

• Cross-reactivity with related proteins:

  • Solution: Validate antibody specificity using peptide competition assays or knockout/knockdown controls. For NB100-93472, blocking with the immunizing peptide confirms specificity .

• Inability to detect surface KIAA0319:

  • Solution: For plasma membrane localization studies, use non-permeabilized immunofluorescence conditions with antibodies targeting the extracellular N-terminus, such as R2 antiserum .

• Difficulty detecting endogenous protein:

  • Solution: Use tissues with known high expression (e.g., brain tissue for KIAA0319) and sensitive detection methods like chemiluminescence for Western blot .

How can I resolve contradictory results when using different KIAA0319 antibodies?

When facing contradictory results with different antibodies:

• Epitope mapping: Determine which regions of KIAA0319 each antibody recognizes. Antibodies targeting different domains may give different results if certain domains are masked in protein complexes or affected by post-translational modifications.

• Isoform specificity: Verify whether the antibodies recognize different KIAA0319 isoforms. For example, antibodies targeting the transmembrane domain will only detect the full-length protein (KA), not the KB or KC variants .

• Antibody validation: Rigorously validate each antibody using knockout/knockdown controls, peptide competition assays, or recombinant expression systems.

• Technical replication: Repeat experiments using standardized protocols to ensure consistent results.

• Combined approach: Use multiple techniques (e.g., Western blot, immunofluorescence, ELISA) to build a comprehensive picture. For instance, antibodies that work well in Western blot may not perform optimally in immunohistochemistry.

• Expert consultation: Consult with researchers experienced in KIAA0319 studies or contact antibody manufacturers for technical support regarding specific applications.

How can KIAA0319 antibodies be utilized to study the protein's role in neurodevelopmental disorders?

KIAA0319 antibodies can be valuable tools for investigating neurodevelopmental disorders:

• Expression pattern analysis: Use immunohistochemistry to map KIAA0319 expression in brain regions relevant to reading and language processing in both normal and dyslexic brain samples.

• Developmental studies: Track KIAA0319 expression during critical periods of neurodevelopment using immunostaining in embryonic and postnatal brain tissues.

• Cell migration assays: Combine KIAA0319 antibodies with neuronal migration markers to investigate how KIAA0319 variants affect cortical development.

• Protein interaction studies: Use co-immunoprecipitation with KIAA0319 antibodies to identify binding partners that may provide insight into molecular pathways affected in dyslexia.

• Functional studies: Combine antibody blocking experiments with neuronal function assays to determine how KIAA0319 affects neuronal connectivity and activity.

What methodological approaches can enhance the specificity and sensitivity of KIAA0319 detection in complex neural tissues?

To improve KIAA0319 detection in neural tissues:

• Antigen retrieval optimization: For fixed tissues, test different antigen retrieval methods (heat-induced versus enzymatic) to maximize epitope accessibility while preserving tissue morphology.

• Signal amplification: Consider using tyramide signal amplification or polymer detection systems for immunohistochemistry to enhance sensitivity without increasing background.

• Dual antibody approaches: Use pairs of antibodies targeting different epitopes of KIAA0319 with distinct fluorophores; co-localization indicates higher specificity.

• Combined IF/FISH techniques: Pair KIAA0319 protein detection with fluorescent in-situ hybridization for KIAA0319 mRNA to correlate protein expression with transcription.

• Tissue clearing methods: Employ tissue clearing techniques (CLARITY, iDISCO) compatible with immunostaining to achieve deeper imaging of KIAA0319 in intact brain circuits.

• Super-resolution microscopy: Use techniques like STORM or STED microscopy for nanoscale localization of KIAA0319, particularly at neuronal membrane surfaces and in trafficking vesicles.