KIFAP3 Antibody

What is KIFAP3 and what is its biological function?

KIFAP3 (Kinesin-associated protein 3), also known as KIF3AP or KAP3, is a protein that binds to the tail domain of KIF3A/3B heterodimers to form a heterotrimeric KIF3 complex. It plays a critical role in regulating the binding of KIF3A/3B to cargo molecules, thereby facilitating intracellular transport processes. KIFAP3 is involved in tethering chromosomes to the spindle pole during cell division and in chromosome movement. At the molecular level, it consists of 792 amino acids with a calculated molecular weight of 91 kDa, though it typically appears as a 91-100 kDa band in Western blots . Recent research has revealed its interactions with proteins involved in cancer progression such as APC and BRK, suggesting its potential role in tumorigenesis and cellular regulation pathways .

What types of KIFAP3 antibodies are available for research applications?

Several KIFAP3 antibodies have been developed for research purposes, predominantly rabbit polyclonal antibodies. Key examples include:

These antibodies recognize different epitopes of KIFAP3 and have been validated for various experimental applications across multiple species .

What are the optimal dilutions and conditions for using KIFAP3 antibodies in Western blotting?

For Western blotting applications using KIFAP3 antibodies, the following methodological considerations are recommended:

For optimal results, proteins should be separated on an 8-10% SDS-PAGE gel due to the relatively high molecular weight of KIFAP3. When performing Western blot analysis, ensure complete transfer of high molecular weight proteins by using appropriate transfer conditions (longer transfer time or lower amperage). For detection, both chemiluminescence (ECL) and fluorescence-based methods have been successfully used, with exposure times of approximately 5 seconds reported for standard ECL detection systems .

How should KIFAP3 antibodies be used for immunohistochemistry (IHC) studies?

For immunohistochemical applications, KIFAP3 antibodies should be used at dilutions ranging from 1:50 to 1:500, with optimization recommended for each specific tissue type and fixation method. For antibody 12700-1-AP, antigen retrieval using TE buffer (pH 9.0) is suggested, though citrate buffer (pH 6.0) may be used as an alternative .

Research has shown successful IHC staining in human ovary cancer tissue and breast cancer tissue microarrays. When performing IHC on breast cancer samples, researchers have used a 1:15 dilution of the HPA023738 antibody for optimal results . Positive staining of KIFAP3 has been observed in 84% (240/285) of breast cancer samples, demonstrating the utility of KIFAP3 as a potential biomarker in cancer research .

What protocols are recommended for KIFAP3 immunoprecipitation (IP) experiments?

For immunoprecipitation of KIFAP3, the following methodology is recommended:

• Use 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate when using antibody 12700-1-AP

• For antibody 164351, a dilution of 1:50-1:100 is recommended

• Positive IP results have been demonstrated in mouse testis tissue with antibody 12700-1-AP

• For cell line experiments, successful IP has been reported using 3 μg of KIFAP3 antibody with 200 μg of HeLa cell extract

When performing co-immunoprecipitation experiments to study KIFAP3 interactions, researchers have successfully demonstrated its association with proteins such as FLA10 (KIF3A homolog) but not with FLA8 (KIF3B homolog), indicating specificity in its binding partners within the kinesin-II complex .

What are the common challenges when detecting KIFAP3 in different sample types?

Detection of KIFAP3 can present several challenges depending on the sample type and experimental conditions:

• Tissue-specific expression levels: KIFAP3 expression varies significantly between tissues, with higher expression typically observed in brain and testis tissues compared to other tissue types . Researchers should adjust antibody concentrations accordingly.

• Sample preparation: Complete cell lysis is crucial for releasing KIFAP3, particularly when working with brain samples where protein extraction can be challenging. Using RIPA buffer supplemented with protease inhibitors is recommended.

• Antibody specificity: Some antibodies may cross-react with other members of the kinesin-associated protein family. Validation using positive control samples (e.g., brain tissue) and negative controls is essential.

• Molecular weight variation: While the calculated molecular weight of KIFAP3 is 91 kDa, it may appear as bands between 91-100 kDa depending on post-translational modifications and experimental conditions .

For optimal results, researchers should first validate the antibody in a system with known KIFAP3 expression and optimize protocols for their specific sample types.

How can KIFAP3 expression levels be accurately quantified by Western blotting and real-time PCR?

For accurate quantification of KIFAP3 expression:

Western Blotting Quantification:

• Include a loading control (β-actin, GAPDH, or α-tubulin) to normalize KIFAP3 expression

• Use a dilution series of a positive control sample to ensure detection is within the linear range

• For densitometric analysis, background-subtracted band intensities should be normalized to loading controls

• When comparing expression between genotypes (e.g., CC vs. TT for rs1541160), a 69.8% decrease in KIFAP3 protein has been observed in CC samples compared to TT samples

Real-time PCR Quantification:

• Use validated KIFAP3 primers with confirmed specificity

• Include multiple reference genes for accurate normalization

• Employ the 2^(-ΔΔCt) method for relative quantification

• In studies comparing KIFAP3 expression between rs1541160 genotypes, expression in CC genotypes was 31.9% less than in TT genotypes in lymphoblastoid cell lines and 41.1% less in brain samples

The consistency between protein reduction (69.8%) and mRNA reduction (31.9-41.1%) suggests post-transcriptional regulation may also play a role in KIFAP3 expression levels .

How does KIFAP3 expression correlate with disease states, particularly in cancer and neurological disorders?

KIFAP3 expression has significant correlations with multiple disease states:

Cancer Associations:

• KIFAP3 is overexpressed in 84% (240/285) of breast cancer samples, making it a potential biomarker

• Its interaction with BRK (Breast tumor kinase) suggests a potential role in breast tumor progression mechanisms

• KIFAP3 also interacts with APC (Adenomatous polyposis coli), a tumor suppressor implicated in colorectal cancer, indicating possible involvement in multiple cancer types

Neurological Disorder Associations:

• Reduced KIFAP3 expression correlates with increased survival in amyotrophic lateral sclerosis (ALS)

• Individuals homozygous for the C allele at SNP rs1541160 show reduced KIFAP3 expression and experience a 14.0-month survival advantage in sporadic ALS compared to those with the TT genotype

• These findings suggest that KIFAP3 may influence motor neuron viability through its role in cellular transport processes

When interpreting KIFAP3 expression data, researchers should consider tissue type, disease context, and genetic background (particularly rs1541160 and rs522444 genotypes) as these factors significantly impact expression levels and potential disease relevance.

What is the significance of KIFAP3 in ciliary function and intraflagellar transport (IFT)?

KIFAP3 plays a crucial role in ciliary function and intraflagellar transport through its association with kinesin-II motors:

• As part of the heterotrimeric kinesin-II complex, KIFAP3 facilitates anterograde transport of cargo proteins along the ciliary axoneme

• The heterodimeric motor organization of kinesin-II, which includes KIF3A/KIF3B (FLA10/FLA8 homologs) and KIFAP3 (KAP), is essential for proper ciliogenesis

• KIFAP3 appears to interact specifically with KIF3A (FLA10) but not with KIF3B (FLA8), suggesting an asymmetric association within the kinesin-II complex that may be critical for proper motor function

• Disruption of KIFAP3 function likely impairs intraflagellar transport, which can lead to ciliopathies - a diverse group of disorders resulting from abnormal ciliary structure or function

This cilia-related function may partially explain KIFAP3's role in neurological disorders like ALS, as neurons rely heavily on intracellular transport mechanisms for survival and function.

How do genetic variants in KIFAP3 affect protein expression and disease outcomes?

Genetic variations in KIFAP3 have significant implications for protein expression and disease outcomes:

SNP rs1541160:

• Located within the KIFAP3 gene

• CC genotype associated with 31.9% reduced KIFAP3 expression in lymphoblastoid cell lines and 41.1% reduced expression in brain samples compared to TT genotype

• Homozygosity for the C allele confers a 14.0-month survival advantage in sporadic ALS patients

• Achieved genome-wide significance (P = 1.84 × 10^-8) in ALS survival analysis

SNP rs522444:

• Located within the KIFAP3 promoter region

• In linkage disequilibrium with rs1541160

• The favorable allele correlates with reduced KIFAP3 expression

Sequence analysis of the KIFAP3 coding region and exon/intron boundaries in individuals with different rs1541160 genotypes did not reveal variants that could explain expression differences, suggesting that these SNPs likely affect transcriptional regulation rather than protein structure .

These findings highlight the importance of considering genetic background when studying KIFAP3 function and suggest that strategies to modulate KIFAP3 expression could potentially influence disease progression in ALS and other conditions where KIFAP3 plays a role.

What are the latest methodological advances for studying KIFAP3 interactions with kinesin motors and other binding partners?

Recent methodological advances for studying KIFAP3 interactions include:

• Co-immunoprecipitation with tagged proteins:

  • Expressing KIFAP3 with tags (GFP, MBP, RFP) alongside potential interaction partners to identify specific binding relationships

  • This approach has revealed that KIFAP3 (KAP) interacts with FLA10 (KIF3A homolog) but not with FLA8 (KIF3B homolog)

• Protein pull-down assays:

  • Using nickel column pull-down for His-tagged proteins to isolate protein complexes

  • The combination of multiple tags (e.g., KAP-GFP-His with FLA10-MBP) allows for sequential purification steps to ensure specificity

• Real-time visualization techniques:

  • Live cell imaging of fluorescently tagged KIFAP3 to track its movement and association with kinesin motors

  • FRAP (Fluorescence Recovery After Photobleaching) analysis to measure dynamics of KIFAP3 association with cilia and cellular structures

• Proximity ligation assays:

  • Detecting protein-protein interactions with high sensitivity in fixed cells and tissues

  • Particularly useful for studying KIFAP3 interactions in their native cellular context

These methods can be employed to further elucidate KIFAP3's role in molecular transport, cell division, and disease mechanisms, potentially identifying new therapeutic targets for conditions where KIFAP3 dysfunction contributes to pathology.