KIF23 Antibody

What is KIF23 and what is its biological significance?

KIF23, also known as MKLP1 (Mitotic Kinesin-Like Protein 1), is a kinesin-like motor protein that plays a critical role in cytokinesis. In humans, the canonical protein consists of 960 amino acid residues with a molecular mass of approximately 110.1 kDa . It is a key component of the centralspindlin complex that mediates microtubule-dependent and Rho-mediated signaling required for myosin contractile ring formation during the cell cycle cytokinesis . KIF23 is primarily localized in both the nucleus and cytoplasm, and its expression has been observed across various tissue types . Its importance extends beyond normal cellular function, as it has been found to be upregulated in several cancer types, including hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC) .

What are the different isoforms of KIF23 and how do they differ structurally?

There are up to three different isoforms of KIF23 reported, with two main splice variants being the most well-characterized:

• KIF23 V1 (Variant 1): The full-length variant containing all exons, including exon 18 which encodes an F-actin interacting domain. This domain may be essential for specific functions unique to the V1 isoform .

• KIF23 V2 (Variant 2): A truncated isoform that lacks exon 18. The nucleotide sequence encoding KIF23 V2 is identical to that of KIF23 V1 except for the absence of exon 18 .

These structural differences suggest potentially distinct functional roles for each variant in cellular processes, particularly in interaction with the actin cytoskeleton .

What are the common applications for KIF23 antibodies in research?

KIF23 antibodies are utilized in multiple research applications:

These applications enable researchers to investigate KIF23 expression, localization, interactions, and functions in various experimental contexts .

How can I validate the specificity of a KIF23 antibody for my research?

To validate KIF23 antibody specificity, implement the following methodological approaches:

• siRNA knockdown experiments: Transfect cells with KIF23-specific siRNAs and confirm reduced antibody signal by Western blotting or immunofluorescence compared to control siRNA-treated cells . This was successfully demonstrated with HLE cells in published research, where KIF23 V1 siRNA treatment significantly reduced the band detected by anti-KIF23 V1 antibody .

• Overexpression studies: Transiently transfect cells with KIF23 V1 or V2 expression constructs and confirm detection of the overexpressed protein with the expected molecular weight . Researchers have validated antibody specificity by overexpressing FLAG-tagged KIF23 variants in HEK293T cells and confirming detection patterns .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples. A specific antibody will show diminished or absent signal when the peptide blocks its binding site.

• Correlation with mRNA expression: Compare antibody staining patterns with RT-PCR results from the same samples to ensure concordance between protein and mRNA expression patterns .

• Testing across multiple cell lines: Confirm consistent detection patterns across cell lines known to express KIF23, such as HLE, Huh7, HepG2, SMMC-7721, and BEL-7402 .

What controls should I include when working with KIF23 antibodies?

Robust experimental design requires appropriate controls:

• Positive tissue controls: Include tissues known to express KIF23, such as proliferating cancer tissues. Hepatocellular carcinoma samples have shown high KIF23 expression and can serve as positive controls .

• Negative controls:

  • Primary antibody omission control

  • Isotype control (using non-specific IgG of the same species and concentration)

  • Tissues known to have low KIF23 expression

• Loading controls: For Western blots, include housekeeping proteins like tubulin or lamin B1, which have been used in KIF23 studies .

• Isoform-specific controls: When studying specific variants, include samples expressing only one variant as demonstrated in studies using transfected HEK293T cells expressing either KIF23 V1 or V2 .

• Subcellular fraction controls: Since KIF23 localizes to both nucleus and cytoplasm, include markers for these compartments when performing subcellular fractionation experiments .

How do I optimize immunohistochemistry protocols for KIF23 detection in tissue samples?

For optimal KIF23 detection in tissue samples by immunohistochemistry:

• Antigen retrieval optimization: KIF23 epitopes may require specific antigen retrieval methods. Published protocols have successfully used heat-induced epitope retrieval in citrate buffer (pH 6.0) .

• Antibody dilution: Determine the optimal antibody concentration through titration experiments (typically starting with manufacturer recommendations and testing 2-fold dilutions). Commercial anti-KIF23 antibodies have been effectively used at dilutions specified in their accompanying documentation .

• Incubation conditions: Overnight incubation at 4°C with primary antibody has yielded good results in published studies, followed by incubation with HRP-conjugated secondary antibodies .

• Detection system selection: The Dako REAL EnVision detection system has been successfully employed for visualizing KIF23 expression in paraffin-embedded tissues .

• Counterstaining and quantification: Use appropriate counterstaining methods and quantify staining using digital microscopy tools as demonstrated in studies using the Olympus CX31 digital microscope for quantifying KIF23-positive cells .

How can I differentiate between KIF23 V1 and V2 isoforms in my experiments?

Distinguishing between KIF23 isoforms requires specific methodological approaches:

• Isoform-specific antibodies: Use antibodies that specifically recognize KIF23 V1, such as those generated against peptides encompassing residues 747-761 of human KIF23 V1 (encoded by exon 18), which is absent in KIF23 V2 . Commercial antibodies that recognize both isoforms can be used alongside variant-specific antibodies for comparative analysis .

• RT-PCR with variant-specific primers: Design primers that specifically amplify each variant. For KIF23 V1, primers targeting exon 18 can be used (e.g., 5'-CAGATTTCCAACGGCCAGCA-3' and 5'-TCATGGCTTTTTGCGCTTGG-3'), while KIF23 V2 can be detected using primers flanking the exon 18 region (e.g., 5'-TCCATCACCTGTGCCTTTACT-3' and 5'-TGGGACTGTCAGTTCATGGC-3') .

• Western blotting: The two isoforms can be distinguished by their molecular weights, with KIF23 V1 appearing as a slightly larger band than KIF23 V2 .

• Expression constructs: Generate constructs expressing only KIF23 V1 or V2 for use as positive controls in your experiments, as demonstrated in studies that created pRK-FLAG-KIF23 V1 and pRK-FLAG-KIF23 V2 expression vectors .

What methodological approaches can be used to study KIF23's role in cancer progression?

To investigate KIF23's involvement in cancer progression:

• Clinical correlation studies: Analyze KIF23 expression in patient samples using immunohistochemistry and correlate with clinical parameters such as tumor stage, metastasis, and patient survival .

• Functional knockdown experiments: Use siRNA or CRISPR-Cas9 to suppress KIF23 expression in cancer cell lines and assess effects on proliferation, migration, invasion, and colony formation capabilities.

• Overexpression studies: Transfect cancer cells with KIF23 variants and compare phenotypic changes, particularly focusing on cell division abnormalities and multinucleation .

• Isoform-specific functional analysis: Compare the effects of KIF23 V1 versus V2 overexpression or knockdown to determine variant-specific contributions to cancer cell phenotypes.

• Live cell imaging: Monitor cytokinesis defects in real-time using fluorescently labeled KIF23 to understand its dynamic behavior during cell division in cancer cells.

• Co-immunoprecipitation: Identify KIF23 interaction partners in cancer cells that might contribute to its role in tumor progression.

How can post-translational modifications of KIF23 be investigated?

To study post-translational modifications (PTMs) of KIF23, particularly ubiquitination which has been documented :

• Immunoprecipitation followed by Western blotting: Precipitate KIF23 and probe with anti-ubiquitin antibodies to detect ubiquitinated forms.

• Mass spectrometry analysis: Perform liquid chromatography-mass spectrometry (LC-MS/MS) on immunoprecipitated KIF23 to identify and map specific modification sites.

• Phosphorylation-specific antibodies: Use commercial phospho-specific antibodies if available, or generate custom antibodies against predicted phosphorylation sites on KIF23.

• In vitro modification assays: Reconstitute potential modification reactions using purified KIF23 and relevant enzymes to characterize the biochemistry of these modifications.

• Mutational analysis: Create KIF23 constructs with mutations at potential modification sites and assess functional consequences in cellular assays.

What are common problems when using KIF23 antibodies for immunohistochemistry and how can they be addressed?

Common challenges and solutions:

• High background staining:

  • Increase blocking time or concentration of blocking agent

  • Reduce primary antibody concentration

  • Perform additional washing steps

  • Use more specific secondary antibodies

• Weak or absent signal:

  • Optimize antigen retrieval conditions

  • Increase antibody concentration or incubation time

  • Use signal amplification systems

  • Ensure proper tissue fixation conditions that preserve KIF23 epitopes

• Non-specific binding:

  • Use additional blocking agents like bovine serum albumin

  • Pre-absorb the antibody with non-specific proteins

  • Test a different antibody clone or manufacturer

• Variable staining across tissue sections:

  • Ensure uniform section thickness

  • Control incubation temperature carefully

  • Use automated staining systems for consistency

How can I accurately quantify KIF23 expression levels in tissue samples?

For reliable quantification of KIF23 expression:

• Digital image analysis: Use software tools to quantify staining intensity and percentage of positive cells, as demonstrated in studies using digital microscopy for assessment .

• Scoring systems: Develop and validate a scoring system that incorporates both staining intensity and percentage of positive cells, which can be particularly useful for correlating expression with clinical outcomes.

• Normalization strategies:

  • Use internal reference genes/proteins when performing RT-PCR or Western blotting

  • Include calibration controls on each slide for immunohistochemistry

  • Process all samples simultaneously when possible to minimize technical variation

• Multiple detection methods: Confirm findings using complementary approaches (e.g., validate IHC results with Western blotting or RT-PCR from the same samples) .

What considerations are important when studying KIF23 in different cell cycle phases?

Since KIF23 functions primarily during cell division:

• Cell synchronization: Use methods like double thymidine block, nocodazole treatment, or serum starvation/refeeding to enrich for cells in specific cell cycle phases.

• Co-staining strategies: Combine KIF23 antibodies with markers for specific cell cycle phases (e.g., phospho-histone H3 for mitosis) to correlate KIF23 localization with cell cycle progression.

• Live cell imaging: For dynamic studies, consider using fluorescently tagged KIF23 constructs to monitor its localization and behavior throughout the cell cycle.

• Flow cytometry: Combine KIF23 staining with DNA content analysis to quantify expression levels across cell cycle phases in large cell populations.

• Fixation timing: When studying highly dynamic processes like cytokinesis, precise timing of fixation is critical to capture KIF23 at centralspindlin complexes.

What emerging technologies could enhance KIF23 research?

Several cutting-edge approaches could advance KIF23 research:

• Single-cell analysis: Apply single-cell RNA sequencing or proteomics to understand cell-to-cell variation in KIF23 expression and function, particularly in heterogeneous tumor samples.

• CRISPR-Cas9 genome editing: Generate knock-in cell lines expressing tagged KIF23 at endogenous levels to study its native dynamics without overexpression artifacts.

• Super-resolution microscopy: Employ techniques like STORM or PALM to visualize KIF23 localization at the centralspindlin complex with nanometer precision.

• Proximity labeling methods: Use BioID or APEX2 fused to KIF23 to identify proximal proteins in living cells, potentially revealing new interaction partners.

• Cryo-electron microscopy: Determine high-resolution structures of KIF23 in complex with its binding partners to understand mechanistic details of its motor function.

How can comparative studies of KIF23 orthologs inform human research?

KIF23 orthologs have been reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken species , offering opportunities for comparative studies:

• Evolutionary conservation analysis: Identify highly conserved domains across species to pinpoint functionally critical regions of the protein.

• Model organism studies: Leverage genetic manipulation in model organisms like zebrafish or mice to understand KIF23 function in development and disease.

• Cross-species antibody validation: Test human KIF23 antibodies for cross-reactivity with orthologs to enable comparative studies across species.

• Tissue-specific expression patterns: Compare expression profiles across species to identify conserved regulatory mechanisms governing KIF23 expression.

• Disease models: Utilize animal models of anemia or cancer to study the role of KIF23 in pathological conditions in vivo.

What are reliable resources for KIF23 research protocols?

For researchers new to KIF23 studies:

• Published methodologies: The immunohistochemistry, Western blotting, and RT-PCR protocols described in peer-reviewed literature provide validated starting points for experimental design .

• Antibody validation resources: Commercial antibody providers often share validation data and recommended protocols specific to their KIF23 antibodies .

• Cell line databases: Resources documenting KIF23 expression levels across cell lines can help select appropriate experimental models.

• Knockout/knockdown validation: Published siRNA sequences effective for KIF23 knockdown provide valuable tools for functional studies .

• Tissue expression databases: Public repositories containing expression data across normal and pathological tissues can inform experimental design and interpretation.