KIF26B Antibody

What is KIF26B and what are its known biological functions?

KIF26B is a highly conserved atypical kinesin belonging to the Kinesin-11 family, composed of 2108 amino acids with a molecular weight of approximately 224 kDa . As a molecular motor protein, KIF26B binds to microtubules and exhibits ATPase activity . It plays critical roles in intracellular transport processes, particularly in the movement of organelles and vesicles within cells .

Research has demonstrated that KIF26B is essential for embryonic development, participating specifically in the development of limbs, face, and kidneys . In kidney development, KIF26B is crucial for maintaining the compact adhesion between mesenchymal cells adjacent to ureteric buds, potentially through interactions with MYH10 . This interaction contributes to establishing the basolateral integrity of the mesenchyme and the polarized expression of ITGA8, which maintains GDNF expression required for further ureteric bud attraction .

What are the common applications for KIF26B antibodies in research?

KIF26B antibodies are versatile tools employed in various molecular and cellular biology techniques. Based on the available data, KIF26B antibodies are primarily used in the following applications:

• Western Blot (WB): For detecting denatured KIF26B protein in cell or tissue lysates, typically revealing bands at approximately 220 kDa .

• Immunohistochemistry (IHC): For visualizing KIF26B expression patterns in paraffin-embedded or frozen tissue sections .

• Immunofluorescence/Immunocytochemistry (IF/ICC): For examining subcellular localization of KIF26B in fixed cells .

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of KIF26B in solution .

These techniques enable researchers to investigate KIF26B expression levels, protein-protein interactions, and localization patterns in various experimental contexts.

What species reactivity is available for commercial KIF26B antibodies?

Currently available KIF26B antibodies demonstrate reactivity across multiple species. The primary species reactivity includes:

• Human

• Mouse

• Rat

Some antibodies may also have predicted reactivity with additional species, including:

• Pig

• Bovine

• Horse

• Sheep

• Rabbit

• Dog

• Chicken

When selecting a KIF26B antibody for your research, it's essential to verify the specific reactivity profile of each antibody to ensure compatibility with your experimental model system.

How is KIF26B expression regulated by signaling pathways such as Wnt5a-Ror?

Recent research has revealed a significant regulatory relationship between the Wnt5a-Ror signaling pathway and KIF26B expression. Mass spectrometry screening identified KIF26B as a high-confidence candidate target of Wnt5a-Ror signaling, with eight unique phosphopeptides from KIF26B showing increased abundance following genetic ablation of Ror expression .

Further validation using specific anti-KIF26B antibodies demonstrated that Kif26b protein levels were elevated in Ror1f/f; Ror2f/f; CAG-CreER MEFs treated with 4-OHT compared to vehicle-treated controls . This observation indicates that Wnt5a-Ror signaling negatively regulates total KIF26B protein expression rather than selectively catalyzing the dephosphorylation of specific sites .

This regulatory mechanism suggests that KIF26B functions as a downstream effector of noncanonical Wnt signaling, potentially mediating its effects on cell motility, polarity, and developmental processes. Researchers investigating KIF26B should consider this regulatory relationship when designing experiments, particularly in contexts where Wnt signaling is active.

What is the role of KIF26B in cancer progression and metastasis?

Emerging evidence suggests that KIF26B contributes to tumorigenesis and malignant behavior in several cancer types. Studies have shown that knockdown of KIF26B inhibits the proliferation and invasion of breast cancer cells, indicating its potential role in promoting cancer progression .

In medulloblastoma, KIF26B has been found to be overexpressed in patient tissues, and this overexpression is associated with poor prognosis . Research examining the molecular mechanisms has revealed that KIF26B may influence medulloblastoma cell proliferation, migration, and cell cycle progression through the PI3K/AKT signaling pathway .

These findings suggest that KIF26B could serve as both a biomarker for cancer diagnosis and prognosis, as well as a potential therapeutic target. Researchers studying KIF26B in the context of cancer should consider examining:

• Correlation between KIF26B expression levels and clinical outcomes

• Effects of KIF26B modulation on cancer cell phenotypes

• Downstream signaling pathways affected by KIF26B expression

• Potential for developing targeted therapies against KIF26B or its associated pathways

What are the best practices for validating KIF26B antibody specificity?

Proper validation of KIF26B antibody specificity is crucial for ensuring reliable research results. Based on established methodologies, the following approach is recommended:

• Western blot validation: Compare protein extracts from wild-type cells with those from KIF26B knockdown or knockout models. A specific KIF26B antibody should detect a band at approximately 220 kDa in wild-type samples but not in knockout samples .

• Multiple antibody validation: Use at least two different antibodies raised against distinct epitopes of KIF26B. In published research, antibodies like 980P and 981P have been used to confirm specific detection of KIF26B protein .

• Knockdown controls: Include shRNA-mediated knockdown controls alongside negative controls (empty vector or non-targeting shRNA) to demonstrate specificity .

• Loading controls: Always use appropriate loading controls such as α-tubulin to ensure equal protein loading across samples .

• Species validation: If working across different species, verify the antibody's reactivity with the KIF26B protein from each species of interest.

Researchers have successfully validated anti-KIF26B antibodies using primary MEFs derived from E12.5 wild-type mice compared with MEFs expressing shRNAs against KIF26B or derived from KIF26B knockout mice .

What are the recommended dilutions and protocols for using KIF26B antibodies in different applications?

While optimal dilutions should be determined by each end-user for their specific experimental conditions, the following general guidelines can be considered for various applications:

For Western Blot (WB):

• Use for detection of denatured KIF26B protein samples

• Expected molecular weight: approximately 224 kDa

• Follow standard Western blot protocols with appropriate blocking and incubation times

For Immunohistochemistry (IHC):

• Applicable for both paraffin-embedded sections (IHC-p) and frozen sections (IHC-f)

• Follow standard immunohistochemical protocols with appropriate antigen retrieval methods

For Immunofluorescence/Immunocytochemistry (IF/ICC):

• Use for subcellular localization studies in fixed cell samples

• Follow standard immunofluorescence protocols with appropriate fixation methods

For ELISA:

• Primarily used for detection of antigenic peptides

• Follow standard ELISA protocols with appropriate blocking and washing steps

It's important to note that the storage conditions for KIF26B antibodies typically involve:

• Storage buffer: PBS with 0.02% Sodium Azide, 50% Glycerol, pH 7.3

• Storage temperature: -20°C

• Avoid freeze/thaw cycles to maintain antibody performance

How can researchers address inconsistent results when detecting KIF26B with antibodies?

Inconsistent results when detecting KIF26B can stem from various factors. Here are methodological approaches to troubleshoot common issues:

• High background signal:

  • Increase the dilution of the primary antibody

  • Extend washing steps between antibody incubations

  • Use a different blocking agent (e.g., switch between BSA and non-fat dry milk)

  • Ensure sample preparation minimizes non-specific binding

• Weak or no signal:

  • Verify KIF26B expression in your experimental system

  • Reduce antibody dilution

  • Extend primary antibody incubation time (e.g., overnight at 4°C)

  • Optimize protein extraction method to ensure KIF26B integrity

  • Consider antigen retrieval methods for IHC applications

• Multiple bands in Western blot:

  • Verify if bands represent known splice variants or post-translational modifications

  • Increase the stringency of washing conditions

  • Use freshly prepared samples to minimize protein degradation

  • Include appropriate controls (KIF26B knockout or knockdown) to identify specific bands

• Conflicting results between different applications:

  • Note that antibody performance can vary between applications (WB, IHC, IF)

  • Some epitopes may be masked in certain applications due to protein folding or fixation

  • Consider using alternative antibodies targeting different epitopes of KIF26B

How should researchers interpret KIF26B expression data in different experimental contexts?

Interpreting KIF26B expression data requires careful consideration of experimental context and controls:

• Developmental studies: KIF26B expression patterns may vary significantly across developmental stages and tissues. When interpreting developmental expression data, consider the temporal and spatial regulation of KIF26B in embryogenesis, particularly in kidney, limb, and facial development .

• Cancer research: In cancer studies, compare KIF26B expression levels between tumor and matched normal tissues. Consider correlations with clinical parameters such as tumor stage, grade, and patient outcomes. The overexpression of KIF26B has been associated with poor prognosis in medulloblastoma, suggesting its potential as a prognostic marker .

• Signaling pathway analysis: When studying KIF26B in the context of signaling pathways such as Wnt5a-Ror, interpret expression changes in relation to pathway activation or inhibition. The negative regulation of KIF26B by Wnt5a-Ror signaling suggests that KIF26B levels may inversely correlate with Wnt pathway activity .

• Functional studies: In knockdown or overexpression experiments, validate the efficiency of genetic manipulation before interpreting phenotypic changes. Correlate KIF26B expression levels with observed cellular phenotypes such as proliferation, migration, or morphological changes.

• Cross-species comparisons: When comparing KIF26B expression across different species, consider the evolutionary conservation of the protein and potential species-specific functions or regulations.

What are the key considerations when studying KIF26B phosphorylation states?

Studying KIF26B phosphorylation requires special attention to several methodological considerations:

• Sample preparation: Phosphorylation states can be labile and easily lost during sample preparation. Use phosphatase inhibitors in all buffers and maintain samples at cold temperatures throughout processing.

• Detection methods: Consider using:

  • Phospho-specific antibodies if available for known phosphorylation sites

  • Phos-tag gels to separate phosphorylated from non-phosphorylated forms

  • Mass spectrometry for identification of phosphorylation sites

• Validation approaches: Research has identified at least fifteen unique phosphopeptides in KIF26B that are regulated by Wnt5a-Ror signaling . When analyzing phosphorylation:

  • Compare phosphorylation levels across multiple experimental conditions

  • Include appropriate controls for phosphatase activity

  • Consider the functional significance of specific phosphorylation sites

• Signaling context: The phosphorylation state of KIF26B appears to be regulated by the Wnt5a-Ror signaling pathway, with significant changes in phosphopeptide abundance upon Ror depletion . Consider this regulatory relationship when designing and interpreting phosphorylation studies.

• Functional implications: Correlate changes in KIF26B phosphorylation with alterations in protein stability, localization, or function to understand the biological significance of these modifications.