KIAA0319 Antibody, Biotin conjugated

What is KIAA0319L and why is it relevant to neurodevelopmental research?

KIAA0319L is a dyslexia-associated protein that plays critical roles in neural development. It functions as a type-I transmembrane protein with potential involvement in axon guidance through interaction with RTN4R . Research indicates that KIAA0319L regulates cell cycle progression during human neuroepithelial cell development, making it a significant target for developmental neurobiology studies . The protein has been implicated in the transition of SOX10+ neuroepithelial cells to PAX6+ neuronal progenitor cells, suggesting its importance in early neurogenesis . Additionally, KIAA0319L serves as a receptor for adeno-associated virus (AAV) and participates in AAV infection through the endocytosis system, which has implications for viral vector-based research and therapeutic applications .

What are the basic structural and biochemical characteristics of KIAA0319L?

KIAA0319L is a 1049 amino acid protein with a calculated molecular weight of 116 kDa, though Western blot typically detects it at 140-150 kDa due to extensive post-translational glycosylation . The protein contains multiple functional domains, including PKD domains in the ectodomain region that may mediate adhesion between neurons and glial fibers during neuronal migration . This highly glycosylated protein undergoes both N- and O-glycosylation, which contributes to its membrane association and function in cell maintenance and cell-cell signaling in the endocytosis pathway . KIAA0319L is encoded by the KIAA0319L gene (Gene ID: 79932, UniProt ID: Q8IZA0) and has alternative names including KIAA1837 and PP791 .

What are the recommended applications for KIAA0319L antibodies?

KIAA0319L antibodies, including biotin-conjugated variants, are primarily validated for Western blotting (WB) and Enzyme-Linked Immunosorbent Assay (ELISA) applications . For Western blotting, dilutions of 1:5000-1:50000 are typically recommended, though optimal dilution may be sample-dependent . These antibodies have demonstrated reactivity with human samples across multiple cell lines including A549, LNCaP, HeLa, HEK-293, HepG2, K-562, and U2OS cells . When conducting immunofluorescence studies, these antibodies have been successfully employed to examine KIAA0319L's subcellular localization, including its presence in the plasma membrane, endosomes, Golgi, and trans-Golgi network .

How does KIAA0319L regulate neuronal differentiation and migration?

KIAA0319L plays a complex role in neuronal differentiation by regulating the transition between different neural cell states. Studies have shown that KIAA0319L knockdown (KD) significantly suppresses SOX10 expression (a marker of neuroepithelial cells) while increasing PAX6 expression (a marker of neuronal progenitor cells) at days 14 and 21 of neuronal differentiation . This suggests that KIAA0319L is critical for maintaining the neuroepithelial state and regulating the timing of transition to neuronal progenitor cells. Further research indicates that KIAA0319L KD decreases the percentage of proliferating (KI67+) neuronal progenitor cells without inducing apoptosis . Additionally, KIAA0319L KD significantly decreases the expression of markers for intermediate progenitor cells (TBR2), immature neuronal cells (TBR1 and β-Tubulin), and mature neuronal cells (MAP2) at day 28 of differentiation . These findings collectively indicate that KIAA0319L regulates multiple aspects of neuronal development, including cell proliferation, differentiation timing, and potentially migration patterns.

What are the molecular mechanisms through which KIAA0319L influences cellular signaling?

KIAA0319L participates in multiple signaling pathways that are essential for neuronal development. Research suggests that it interacts with the intracellular trafficking protein AP-2, indicating a role in endocytosis-mediated signaling . Additionally, KIAA0319L is associated with extracellular signaling pathways via Smad2/3, which are crucial regulators of neurogenesis . The protein undergoes proteolytic processing, similar to other signaling receptors, which may generate fragments with distinct signaling functions . The presence of PKD domains in the ectodomain region suggests a role in mediating adhesion between neurons and glial fibers during neuronal migration . Furthermore, a secreted, non-membrane minor isoform of KIAA0319L has been detected, pointing toward a potential role in paracrine or autocrine signaling . These diverse signaling mechanisms likely contribute to KIAA0319L's complex functions in neuronal development and dyslexia pathophysiology.

How can researchers optimize immunoprecipitation protocols using biotin-conjugated KIAA0319L antibodies?

For optimal immunoprecipitation using biotin-conjugated KIAA0319L antibodies, researchers should consider several key factors. First, the biotin conjugation provides an advantage for pull-down assays using streptavidin-coated magnetic beads, which offer high affinity and specificity . The recommended protocol involves incubating 2-5 μg of the biotin-conjugated antibody with 50 μl of streptavidin beads for 30 minutes at room temperature, followed by washing with PBS containing 0.01% Tween-20 . When working with KIAA0319L, pre-clearing cell lysates is essential due to the protein's highly glycosylated nature and tendency to participate in multiple protein complexes . Given that KIAA0319L undergoes proteolytic processing, researchers should be aware that immunoprecipitation might pull down fragments of various sizes (ranging from 116 kDa to 150 kDa) . For cross-linking experiments, it's advisable to use DSP (dithiobis(succinimidyl propionate)) at 2 mM concentration to preserve protein-protein interactions while maintaining the integrity of the biotin tag on the antibody . The storage buffer composition (50% glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300) helps maintain antibody stability during the procedure .

What are the optimal conditions for Western blotting with KIAA0319L antibodies?

When performing Western blot analysis with KIAA0319L antibodies, researchers should optimize several critical parameters based on the protein's unique characteristics. Given that KIAA0319L has a predicted molecular weight of 116 kDa but typically appears at 140-150 kDa due to glycosylation , separation should be conducted on lower percentage gels (6-8%) to achieve proper resolution of these high molecular weight species. For more detailed analysis of potential proteolytic fragments, higher concentration gels (10-12% or 4-12% gradient) are recommended . Sample preparation should include protease inhibitors to prevent degradation, and deglycosylation with PNGase F or O-glycosidase can be performed to examine the contribution of glycosylation to band patterns .

The recommended dilution range for Western blotting is 1:5000-1:50000, though this should be optimized for each specific experiment and cell type . Transfer conditions should be adjusted for high molecular weight proteins, typically using lower current (250 mA) for longer duration (90-120 minutes) when transferring to PVDF membranes. When detecting KIAA0319L in complex samples, blocking with 5% non-fat milk in TBST for 1 hour at room temperature helps reduce background, while overnight primary antibody incubation at 4°C improves specific binding .

How can researchers effectively validate KIAA0319L antibody specificity in neurodevelopmental studies?

Validating KIAA0319L antibody specificity is crucial for neurodevelopmental studies due to the protein's complex processing and involvement in multiple cellular pathways. A comprehensive validation approach should include multiple complementary techniques. First, perform side-by-side comparison using both N-terminal and C-terminal targeting antibodies, as KIAA0319L undergoes proteolytic processing that may affect epitope availability . Western blot analysis should demonstrate the expected molecular weight range (140-150 kDa for full-length glycosylated protein) and show reduced or absent signal following KIAA0319L knockdown or knockout .

Immunofluorescence validation should demonstrate the expected subcellular localization patterns including plasma membrane, endosomes, Golgi, and trans-Golgi network . Co-localization studies with established markers for these compartments (e.g., plasma membrane: Na⁺/K⁺-ATPase; Golgi: GM130; endosomes: EEA1) can further confirm specificity . For functional validation, researchers should observe altered expression patterns of downstream markers following KIAA0319L manipulation, such as changes in SOX10, PAX6, TBR2, TBR1, and MAP2 levels in neuronal differentiation models .

For biotin-conjugated antibodies specifically, additional controls should include unconjugated primary antibody and streptavidin-only conditions to confirm that the conjugation process has not altered epitope recognition or introduced non-specific binding .

What considerations are important when designing ELISA protocols with biotin-conjugated KIAA0319L antibodies?

When designing ELISA protocols using biotin-conjugated KIAA0319L antibodies, researchers should optimize several parameters to ensure sensitivity and specificity. The biotin conjugation provides significant advantages for detection in sandwich ELISA formats, allowing for amplification through streptavidin-HRP systems . Initial coating of plates should be performed with a capture antibody targeting a different epitope of KIAA0319L than the biotin-conjugated detection antibody to avoid competitive binding . Due to KIAA0319L's extensive glycosylation, blocking solutions containing 1-2% BSA in PBS are preferred over milk-based blockers, which may interfere with glycoprotein detection .

For quantitative ELISA, a standard curve should be established using recombinant KIAA0319L protein (particularly the immunogen fragment 479-718AA) , with concentrations ranging from 0.1-100 ng/ml. Sample dilutions should be optimized based on the expected KIAA0319L concentration in neuronal cells or brain tissues. When detecting KIAA0319L in complex biological samples, pre-clearing with Protein G can reduce background interference .

To optimize detection, biotin-conjugated antibody dilutions should start at 1:1000 and be titrated to determine optimal signal-to-noise ratio . Development times with streptavidin-HRP should be carefully monitored (typically 10-30 minutes) to prevent oversaturation. For multiplex ELISA applications targeting both KIAA0319L and related developmental markers, careful antibody selection is necessary to avoid cross-reactivity with the related KIAA0319 protein .

How should researchers interpret multiple banding patterns in Western blots with KIAA0319L antibodies?

When analyzing Western blot results for KIAA0319L, researchers frequently encounter multiple bands that require careful interpretation. The expected molecular weight of unmodified KIAA0319L is 116 kDa, but it typically appears as bands at 140-150 kDa due to extensive glycosylation . Previous studies have reported detection of four distinct bands corresponding to monomeric/dimeric and partially/fully glycosylated forms, with apparent sizes around 150, 200, 300, and 400 kDa . Additionally, KIAA0319L undergoes proteolytic processing, which can generate smaller fragments detectable with C-terminal antibodies when separated on higher concentration gels (10-12% or 4-12%) .

To interpret these complex banding patterns, researchers should consider several factors. Multiple high molecular weight bands (>150 kDa) may represent homodimeric conformations that haven't been fully reduced during sample preparation . Bands between 116-150 kDa typically represent differentially glycosylated monomeric forms, which can be confirmed by deglycosylation treatment . Smaller fragments (<116 kDa) likely represent proteolytic products and may provide insights into KIAA0319L processing and signaling . Bands that appear inconsistently across different cell types may reflect tissue-specific post-translational modifications or processing events . For definitive identification of specific bands, comparison with lysates from KIAA0319L knockdown or knockout samples is highly recommended .

What are common challenges in immunofluorescence studies with KIAA0319L antibodies and how can they be addressed?

Immunofluorescence studies with KIAA0319L antibodies present several technical challenges that require specific optimization strategies. One primary challenge is the protein's distribution across multiple subcellular compartments (plasma membrane, endosomes, Golgi, and trans-Golgi network) , which can make interpretation difficult. To address this, co-staining with compartment-specific markers such as Na⁺/K⁺-ATPase (plasma membrane), EEA1 (early endosomes), GM130 (Golgi), or TGN46 (trans-Golgi network) is essential for accurate localization analysis .

Another common issue is detection sensitivity, particularly in cells with lower endogenous expression. This can be mitigated by using tyramide signal amplification systems compatible with biotin-conjugated antibodies . For studies examining KIAA0319L during neuronal differentiation, temporal specificity is critical, as expression patterns change significantly between different developmental stages (days 14, 21, 28, and 42 show distinct patterns) . Researchers should carefully select timepoints based on the developmental process under investigation.

Fixation methods also significantly impact KIAA0319L detection. For membrane-associated KIAA0319L, mild fixation with 2% paraformaldehyde for 10-15 minutes preserves epitope accessibility better than methanol fixation . Background fluorescence, particularly with biotin-conjugated antibodies, can be reduced by including an avidin/biotin blocking step before primary antibody incubation . For detecting low-abundance KIAA0319L in primary neuronal cultures, overnight primary antibody incubation at 4°C followed by extended secondary antibody incubation (2-3 hours) at room temperature improves signal detection .

How can researchers address experimental inconsistencies when studying KIAA0319L in different neural cell populations?

Experimental inconsistencies when studying KIAA0319L across different neural cell populations are common due to the protein's differential expression and processing during development. To address these challenges, researchers should implement several strategies. First, establish baseline expression profiles for KIAA0319L across the specific neural populations under study using quantitative RT-PCR and Western blotting before conducting functional experiments . This baseline characterization helps interpret subsequent experimental outcomes in proper context.

For developmental studies, carefully synchronize cell populations using established protocols, as KIAA0319L expression and function vary significantly between SOX10+ neuroepithelial cells, PAX6+ neuronal progenitors, and more differentiated neuronal populations . When comparing KIAA0319L knockdown effects, confirm consistent knockdown efficiency across different cell types, as variable silencing efficiency can lead to apparently contradictory results .

Post-translational modifications of KIAA0319L, particularly glycosylation patterns, may differ between cell types and developmental stages . To account for this, parallel analysis of glycosylation status using glycosidase treatments can help normalize comparisons . When studying KIAA0319L's role in proliferation, standardize cell cycle phase distribution across experimental groups, as its effects on KI67+ cells may be cell-cycle dependent .

For biotin-conjugated antibody applications specifically, validate consistent antibody performance across different neural populations by testing serial dilutions (1:500, 1:1000, 1:2000) to identify optimal working concentrations for each cell type . Finally, when comparing results across laboratories or studies, consider differences in antibody clone, epitope recognition, and conjugation methods, which may significantly impact detection patterns and experimental outcomes .

How might biotin-conjugated KIAA0319L antibodies facilitate new research into dyslexia pathophysiology?

Biotin-conjugated KIAA0319L antibodies offer significant potential for advancing dyslexia research through several innovative applications. Their high-affinity binding properties combined with biotin-streptavidin detection systems enable enhanced sensitivity for identifying subtle alterations in KIAA0319L expression or localization in dyslexia-relevant brain regions . This could reveal previously undetectable patterns in post-mortem tissue samples from individuals with dyslexia compared to neurotypical controls. The biotin conjugation also facilitates multiplexed immunohistochemistry approaches, allowing simultaneous visualization of KIAA0319L alongside other dyslexia-associated proteins (such as DCDC2, DYX1C1) and neuronal migration markers .

In functional studies, these antibodies can be applied to track KIAA0319L trafficking in real-time using quantum dot-streptavidin conjugates, potentially revealing abnormal protein dynamics in cellular models derived from individuals with dyslexia . For mechanistic investigations, biotin-conjugated antibodies enable more efficient chromatin immunoprecipitation sequencing (ChIP-seq) studies to identify transcriptional networks regulated by KIAA0319L's intracellular domain, which may undergo nuclear translocation following proteolytic processing .

Additionally, the combination of these antibodies with proximity ligation assays (PLA) could identify novel protein interaction partners of KIAA0319L in neural tissue, potentially uncovering dyslexia-specific alterations in signaling networks . This approach might reveal how KIAA0319L interfaces with other dyslexia-associated proteins and whether these interactions are disrupted in dyslexic neural tissue, providing new therapeutic targets for intervention.

What novel methodologies could benefit from the application of KIAA0319L biotin-conjugated antibodies?

Emerging methodologies in neuroscience research could be significantly enhanced through the application of biotin-conjugated KIAA0319L antibodies. Spatial transcriptomics combined with immunofluorescence using these antibodies could map KIAA0319L protein expression alongside its transcriptional landscape in intact brain tissue, revealing region-specific regulation patterns relevant to dyslexia . The biotin conjugation provides excellent compatibility with tissue clearing techniques such as CLARITY and iDISCO, enabling whole-brain imaging of KIAA0319L distribution in animal models with unprecedented resolution .

For single-cell proteomics applications, biotin-conjugated antibodies can be incorporated into cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) protocols, allowing simultaneous measurement of KIAA0319L protein levels and transcriptional profiles in heterogeneous neural populations . In organoid research, these antibodies facilitate live-cell surface labeling of KIAA0319L in cerebral organoids, enabling sorting of specific progenitor populations based on KIAA0319L expression levels for detailed characterization .

The high specificity of biotin-streptavidin interactions also makes these antibodies ideal for super-resolution microscopy techniques like STORM and PALM, potentially revealing nanoscale organization of KIAA0319L in neuronal membranes and its colocalization with synaptic markers . For therapeutic development, biotin-conjugated KIAA0319L antibodies could be used to functionalize nanoparticles for targeted delivery of gene editing machinery to KIAA0319L-expressing cells, opening avenues for precise modification of dyslexia-associated genetic variants .