KIF21B Antibody

What is KIF21B and why is it important in research?

KIF21B is a member of the kinesin superfamily of motor proteins with significant roles in regulating microtubule dynamics. It functions as a processive motor and has been identified as essential for neuronal development and function . KIF21B has gained research importance due to its associations with multiple diseases, including colorectal cancer where it correlates with poor prognosis , neurodevelopmental disorders, multiple sclerosis as a risk gene with central nervous system function , and Alzheimer's disease where expression levels are significantly altered . The protein's critical role in both neurological and immunological contexts makes KIF21B antibodies essential tools for studying these disease mechanisms.

Which cell types express KIF21B and how does this influence antibody selection?

KIF21B is predominantly expressed in neurons and astrocytes but not in microglia cells or SMI32-positive axons . Immunohistochemistry studies have confirmed KIF21B expression in both neurons and glia cells, with particularly high expression in reactive astrocytes . When selecting KIF21B antibodies for research, consideration of cell type-specific expression patterns is crucial. For neuronal studies, antibodies optimized for dendrite detection may be preferred, while astrocyte studies might require antibodies that can distinguish between basal and activated expression states. Validation of antibody specificity across different cell types is essential, as demonstrated in studies using both knockout controls and multiple detection methods.

How do I validate the specificity of a KIF21B antibody?

Validating KIF21B antibody specificity requires a multi-method approach. Western blotting should be performed using both positive controls (tissues/cells known to express KIF21B, such as brain tissue or Jurkat T cells) and negative controls (KIF21B knockout samples) . In published studies, KIF21B-specific antibodies were confirmed using Southern blotting and PCR analysis alongside Western blotting to ensure gene targeting was successful . Immunohistochemical staining comparing wild-type and knockout tissues provided further validation, with knockout tissues showing no detectable KIF21B signal above background levels . For researchers without access to knockout models, siRNA knockdown cells can serve as alternative negative controls, though with potential limitations in complete protein elimination.

What are the optimal methods for KIF21B detection in brain tissue samples?

For KIF21B detection in brain tissue, immunohistochemistry and Western blotting have been successfully employed. When performing immunohistochemistry, researchers should consider age-matched controls since KIF21B expression varies with age, particularly in pathological conditions such as Alzheimer's disease . For Western blotting, protocols have been validated using brain tissue from both wild-type and knockout mice to confirm specificity . When analyzing human brain samples, it's important to account for post-mortem delay (PMD) as a potential confounding factor, though studies have shown KIF21B expression remains relatively stable across different PMD timeframes . For optimal results, semi-quantitative scoring of KIF21B expression should be performed in both neurons and glia cells separately, as these can show differential expression patterns in disease states.

How can I effectively detect KIF21B in immune cells for studying its role in immune synapse formation?

For studying KIF21B in immune cells, particularly T lymphocytes such as Jurkat cells, immunofluorescence techniques combined with functional assays have proven effective . Researchers should first confirm KIF21B expression in their immune cell model via Western blotting using validated antibodies . When studying immune synapse formation, combining anti-CD3 coated surfaces to mimic T-cell activation with phalloidin staining to visualize the peripheral actin ring provides context for KIF21B localization . Live-cell imaging using Differential Interference Contrast (DIC) microscopy can complement fixed-cell approaches to assess dynamic processes. For quantitative analysis, measurements of synapse size and formation kinetics in both wild-type and KIF21B-deficient cells can provide functional insights into KIF21B's role in immune cell biology .

What techniques are recommended for studying KIF21B in colorectal cancer samples?

For colorectal cancer (CRC) research, a combination of bioinformatics analysis and experimental validation approaches is recommended. Studies have utilized multiple databases (UALCAN, GEPIA, TIMER) to analyze KIF21B expression patterns across cancer samples . For experimental validation in CRC cell lines, real-time qPCR and Western blotting have successfully confirmed KIF21B upregulation . When designing functional studies, techniques such as CCK8 assay for cell proliferation, flow cytometry for apoptosis assessment, and transwell assays for migration and invasion measurements have effectively demonstrated the impact of KIF21B deficiency on cancer cell behaviors . Additionally, immunohistochemistry on patient-derived CRC samples can provide clinically relevant data on KIF21B expression patterns that can be correlated with survival data and other clinical parameters.

How does KIF21B expression change across different neurodegenerative conditions, and what antibody considerations apply?

KIF21B expression shows disease-specific patterns across neurodegenerative conditions. In Alzheimer's disease (AD), KIF21B is significantly increased compared to both multiple sclerosis (MS) and normal controls, with particularly pronounced differences in younger patients (below 62 years) . In AD patients under 62 years, KIF21B expression is approximately six-fold higher than in normal controls and five-fold higher than in MS patients . These expression differences must be considered when designing experiments and interpreting results. Age stratification is critical, as KIF21B expression differences between disease groups diminish in older patients (>72 years) . Additionally, researchers should account for potential neuron density changes by including markers such as NeuN (RBFOX3), as significant reductions in neuron density were observed in elderly AD patients despite similar KIF21B expression levels .

What methodological approaches can detect the relationship between KIF21B and astrocyte activation in neurological disorders?

To study KIF21B in astrocyte activation, researchers should employ both in vitro cell culture systems and tissue analysis approaches. Cell culture experiments using astrocytoma cell lines (e.g., U251) or primary astrocytes with inflammatory stimuli (IL-1β and IFN-γ) can demonstrate KIF21B upregulation during activation . When designing such experiments, activation status should be confirmed by measuring inflammatory markers such as IL-6 production . For tissue analysis, double immunostaining for KIF21B and GFAP (astrocyte marker) can identify co-localization patterns. Correlation analysis between KIF21B and GFAP mRNA expression has revealed significant positive correlations in young AD patients and in the white matter of MS patients but not in normal controls, suggesting disease-specific activation patterns . For comparative analysis, other kinesins (e.g., KIF1Bα and KIF5A) should be included as controls, as they show different regulation patterns during astrocyte activation .

How can I design experiments to study the role of KIF21B in microtubule dynamics using antibody-based approaches?

Studying KIF21B's role in microtubule dynamics requires sophisticated experimental approaches that combine genetic manipulation with advanced microscopy. In neuronal systems, KIF21B knockout models have revealed that microtubules grow more slowly and persistently in the absence of KIF21B, leading to tighter packing in dendrites . For experimental design, researchers should consider using live-cell imaging with fluorescently tagged tubulin to monitor microtubule growth rates, catastrophe frequencies, and rescue events in both wild-type and KIF21B-deficient cells. Antibody-based approaches can complement these studies through fixed-cell immunofluorescence, where co-staining for KIF21B and microtubule plus-end tracking proteins (+TIPs) can reveal functional interactions. In immune cells, similar approaches have demonstrated that KIF21B knockout causes microtubule overgrowth and perturbs centrosome translocation , highlighting the importance of cell-type specific experimental designs when studying KIF21B's role in cytoskeletal regulation.

Why might I observe variable KIF21B staining patterns in white matter tissue, and how can I address this?

Variable KIF21B staining in white matter tissue has been documented, with some areas showing high expression and others completely negative within the same tissue sample . This variability likely reflects activation states of cells rather than technical artifacts, as it has been observed consistently across studies. To address this challenge, researchers should implement a systematic sampling approach, examining multiple regions within each white matter sample. Semi-quantitative scoring systems that account for both staining intensity and percentage of positive cells have been successfully employed in previous studies . Additionally, correlation with activation markers can help interpret this variability. For example, in MS patients, GFAP expression correlates with KIF21B expression in white matter, suggesting that astrocyte activation may underlie some of the observed heterogeneity . Researchers should also consider double-labeling approaches to identify which specific cell populations contribute to the variable expression patterns.

How can I address contradictory results between mRNA and protein expression of KIF21B?

When facing discrepancies between KIF21B mRNA and protein expression data, several methodological considerations can help resolve contradictions. First, validate both detection methods using appropriate controls. For mRNA analysis, ensure primer specificity through melt curve analysis and include no-template controls. For protein detection, include both positive and negative controls as previously described. Second, consider post-transcriptional regulation mechanisms that might affect the correlation between mRNA and protein levels. Third, examine cell type-specific expression patterns, as bulk tissue analysis may obscure cell type-specific differences. In previous research, kif21b mRNA findings in grey matter tissues were validated at the protein level through immunohistochemistry, confirming increased expression in AD patients compared to MS and normal controls in both neurons and glia cells . Finally, temporal dynamics should be considered, as mRNA expression changes may precede protein level alterations or vice versa.

What controls should I include when studying KIF21B in genetically modified cell lines or animal models?

When working with genetically modified systems to study KIF21B, comprehensive controls are essential for result interpretation. For CRISPR/Cas9 knockout cell lines, researchers should select and validate multiple independent clones to account for potential off-target effects . Western blotting confirmation of KIF21B absence is crucial, and rescue experiments through re-expression of KIF21B (such as KIF21B-GFP) provide critical functional validation . For animal models, successful gene targeting should be confirmed through multiple methods, including Southern blotting, PCR analysis, Western blotting, and immunohistochemistry . Additionally, researchers should account for potential compensatory mechanisms, particularly from related kinesin family members. Phenotypic characterization should include both cellular and behavioral assessments, as KIF21B-null mice exhibit behavioral changes involving learning and memory deficits that correlate with observed cellular alterations .

How might KIF21B genotype influence antibody selection for personalized medicine applications?

KIF21B genetic variations have been associated with multiple diseases, including neurodevelopmental disorders, ankylosing spondylitis, and ulcerative colitis . These genetic associations suggest that KIF21B genotyping could potentially guide personalized medicine approaches. For research in this area, antibodies that can specifically recognize different KIF21B variants or phosphorylation states may become increasingly important. Development of antibodies that can distinguish between wild-type KIF21B and disease-associated variants would enable more precise characterization of molecular mechanisms underlying these conditions. Current research has included genotype information in study designs, as evidenced by the incorporation of KIF21B genotype data (AA, GA, or GG) in patient cohorts studied for neurodegenerative diseases . Future antibody-based approaches might include proximity ligation assays to detect genotype-specific protein interactions or phospho-specific antibodies to identify altered signaling pathways associated with specific KIF21B variants.

What are the emerging techniques for studying KIF21B in relation to immune cell dysfunction in multiple sclerosis?

Emerging approaches for studying KIF21B in MS may combine single-cell technologies with traditional antibody-based methods. Single-cell RNA-sequencing has already been employed to analyze the functional state of KIF21B across cancer types , and similar approaches could elucidate cell type-specific roles in MS. For immune cell studies, advanced live-cell imaging techniques that capture KIF21B dynamics during immune synapse formation will be valuable, as KIF21B knockout has been shown to affect synapse size and formation kinetics . Additionally, correlative light and electron microscopy could provide unprecedented insights into KIF21B's structural role at the immune synapse. Patient-derived immune cells with different KIF21B risk alleles may reveal genotype-specific functional differences. Researchers should consider how KIF21B interacts with other MS risk genes, potentially through protein-protein interaction studies using co-immunoprecipitation with KIF21B antibodies followed by mass spectrometry analysis to identify novel binding partners in immune cells.

How can KIF21B antibodies be utilized in developing potential therapeutic approaches for colorectal cancer?

Given KIF21B's association with poor prognosis in colorectal cancer , antibody-based approaches may contribute to both diagnostic and therapeutic strategies. For diagnostics, KIF21B antibodies could be employed in immunohistochemical panels to stratify patients based on expression levels, potentially guiding treatment decisions. Development of more sensitive detection methods, such as proximity extension assays or highly sensitive ELISA techniques, might enable KIF21B detection in liquid biopsies. For therapeutic development, researchers could explore antibody-drug conjugates targeting KIF21B, though this would require careful assessment of expression in normal tissues to minimize off-target effects. Alternatively, small molecule inhibitors of KIF21B could be developed, with antibody-based assays serving as critical tools in high-throughput screening platforms. Finally, functional studies have shown that KIF21B deficiency reduces cell proliferation, migration, and invasion in cancer cells , suggesting that targeted downregulation approaches might have therapeutic potential, with antibodies serving as vital tools to monitor treatment efficacy.