KIFAP3 Antibody, HRP conjugated

What is KIFAP3 and why is it significant in research?

KIFAP3, also known as KIF3AP or KAP3, functions as a novel KIF3A/3B-associated protein that binds to the tail domain of KIF3A/3B, potentially regulating their cargo binding capabilities . The KIFAP3 gene is located on chromosome 1q24 and encodes a 90 kDa protein containing three armadillo repeats . Recent studies have highlighted KIFAP3's significance in various conditions, including its overexpression in breast cancer (detected in 84% of cases) and its potential involvement in amyotrophic lateral sclerosis (ALS), making it a valuable research target .

What are the typical applications for KIFAP3 antibodies in laboratory research?

KIFAP3 antibodies have been validated for multiple research applications including Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence (IF/ICC), Immunoprecipitation (IP), and ELISA . WB applications typically employ dilutions ranging from 1:500 to 1:3000, while IHC applications use dilutions between 1:50 and 1:500, depending on the specific antibody and experimental conditions . These applications allow researchers to study KIFAP3 expression patterns, protein interactions, and localization within various tissues and cell types.

What is the significance of HRP conjugation in KIFAP3 antibodies?

HRP (Horseradish Peroxidase) conjugation provides significant advantages for detection systems in numerous applications. While many commercially available KIFAP3 antibodies are unconjugated , HRP-conjugated versions offer enhanced sensitivity and convenience by eliminating the need for secondary antibody incubation steps. The conjugation-ready formats are designed for use with various detection systems, including fluorochromes, metal isotopes, oligonucleotides, and enzymes, making them versatile tools for antibody labeling, functional assays, and multiplex imaging applications .

How should sample preparation be optimized for KIFAP3 detection in different tissue types?

For optimal KIFAP3 detection across different tissue types, sample preparation techniques should be tailored to the specific application. For IHC applications with paraffin-embedded tissues, heat-mediated antigen retrieval using TE buffer (pH 9.0) is recommended, though citrate buffer (pH 6.0) may serve as an alternative . For human tissues such as ovary cancer and testis, specific antibodies have demonstrated reliable results in IHC applications . For protein extraction in WB applications, positive KIFAP3 detection has been confirmed in human brain tissue, testis tissue, as well as cell lines including MCF-7, HeLa, SH-SY5Y, and C6, suggesting these as appropriate positive controls .

What are the optimal protocols for Western blotting with KIFAP3 antibodies?

For Western blot applications using KIFAP3 antibodies, the following protocol elements are recommended:

It is essential to note that KIFAP3 protein typically appears between 91-100 kDa, slightly higher than the calculated molecular weight of 91 kDa .

What considerations should be made when performing immunohistochemistry with HRP-conjugated KIFAP3 antibodies?

When performing IHC with HRP-conjugated KIFAP3 antibodies, researchers should consider optimizing the following parameters:

• Antigen retrieval: TE buffer at pH 9.0 is suggested as the primary choice, with citrate buffer at pH 6.0 as an alternative .

• Antibody dilution: Begin with manufacturer-recommended dilutions (typically 1:20-1:200 for monoclonal or 1:50-1:500 for polyclonal) and optimize for specific tissue types .

• Incubation conditions: The specific temperature and duration should be optimized based on the tissue type and fixation method.

• Detection system: With HRP-conjugated antibodies, a direct detection system can be employed, eliminating the need for secondary antibody incubation.

• Counterstaining: Appropriate counterstaining should be selected based on the localization pattern of KIFAP3, which has been reported in the nucleus, cytoplasm, and endoplasmic reticulum .

How can KIFAP3 antibodies be utilized in studying cancer progression mechanisms?

KIFAP3 antibodies present valuable tools for investigating cancer progression mechanisms, particularly in breast cancer where KIFAP3 overexpression has been documented in 84% (240/285) of cases . Researchers can employ KIFAP3 antibodies in combination with tissue microarrays to evaluate KIFAP3 expression patterns across different cancer subtypes and stages. Additionally, the interaction between KIFAP3 and SmgGDS, which regulates GTP/GDP exchange in small G proteins including members of the Rho family and Ki-Ras, suggests potential involvement in signaling pathways critical to cancer progression . Immunofluorescence studies using KIFAP3 antibodies can help elucidate subcellular localization changes during malignant transformation, while co-immunoprecipitation experiments can identify novel protein interactions in cancer contexts.

What approaches can be used to study KIFAP3 interactions with KIF3A/B using antibody-based techniques?

To study KIFAP3 interactions with KIF3A/B and other binding partners, researchers can implement several antibody-based techniques:

• Co-immunoprecipitation: Using KIFAP3 antibodies (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate) to pull down protein complexes, followed by Western blot analysis for KIF3A/B detection .

• Proximity ligation assays: These can visualize and quantify protein-protein interactions between KIFAP3 and KIF3A/B at the single-molecule level within cells.

• Immunofluorescence co-localization: Dual labeling experiments using KIFAP3 antibodies alongside KIF3A/B-specific antibodies can confirm co-localization patterns, particularly in cell lines such as HepG2 where positive IF/ICC detection has been demonstrated .

• ChIP-seq approaches: These can be employed to investigate potential roles of KIFAP3 in transcriptional regulation, given its reported nuclear localization .

How can researchers troubleshoot cross-reactivity issues with KIFAP3 antibodies in multi-species studies?

Cross-reactivity considerations are crucial when designing multi-species studies with KIFAP3 antibodies. The following strategies can help address potential issues:

• Sequence homology analysis: KIFAP3 antibodies targeting amino acids 683-732 show high sequence homology (100%) across multiple species including human, mouse, rat, bovine, rabbit, horse, pig, and guinea pig, while showing slightly reduced homology in dog (92%) and chicken (85%) .

• Validation controls: Include appropriate positive and negative controls from different species to validate antibody specificity before proceeding with complex experiments.

• Epitope mapping: Understanding the specific epitope recognized by the antibody can help predict potential cross-reactivity issues. KIFAP3 antibodies generated against synthetic peptides located between aa683-732 of human KIFAP3 (Q92845, NP_055785) demonstrate broad cross-species reactivity .

• Pre-absorption controls: These can help determine if observed signals are specific to the intended target or result from non-specific binding.

How should researchers normalize and quantify KIFAP3 expression in Western blot analyses?

For accurate normalization and quantification of KIFAP3 expression in Western blot analyses, researchers should:

• Select appropriate loading controls based on experimental context (e.g., β-actin, GAPDH, or tissue-specific housekeeping proteins).

• Perform densitometric analysis using specialized software to quantify band intensities.

• Calculate the relative expression ratio of KIFAP3 to the loading control.

• Consider using a standard curve of recombinant KIFAP3 protein for absolute quantification when necessary.

• Report observed molecular weight (91-100 kDa) alongside expected molecular weight (91 kDa) to account for post-translational modifications that may affect protein migration .

What are the best practices for interpreting KIFAP3 localization patterns in cellular compartments?

When interpreting KIFAP3 localization patterns using immunofluorescence or immunohistochemistry techniques, researchers should:

• Note that KIFAP3 has been reported to localize to multiple cellular compartments including the nucleus, cytoplasm, and endoplasmic reticulum .

• Use established organelle markers in co-localization studies to confirm compartment-specific localization.

• Consider cell type-specific variations in localization patterns, as KIFAP3 expression and distribution may vary between tissues.

• Evaluate potential changes in localization under different experimental conditions, as protein trafficking may be regulated by cellular stress, cell cycle progression, or disease states.

• Use appropriate microscopy techniques with sufficient resolution to distinguish between closely associated structures.

How can discrepancies in KIFAP3 detection between different antibody clones be reconciled?

When encountering discrepancies in KIFAP3 detection between different antibody clones, researchers should implement the following strategies:

• Compare epitope specificity: Antibodies targeting different regions of KIFAP3 (N-terminal, middle region, C-terminal) may yield different results based on protein folding, complex formation, or post-translational modifications .

• Evaluate antibody validation data: Review validation data from manufacturers, including Western blot images, IHC staining patterns, and specificity testing results .

• Perform parallel testing: Use multiple antibody clones simultaneously under identical experimental conditions to directly compare performance.

• Consider antibody format differences: Monoclonal antibodies (e.g., 60266-1-Ig) may offer higher specificity but potentially lower sensitivity compared to polyclonal antibodies (e.g., 12700-1-AP) .

• Implement alternative detection methods: Validate findings using independent techniques such as mass spectrometry or RNA expression analysis.

How might KIFAP3 antibodies contribute to understanding neurodegenerative disease mechanisms?

KIFAP3 antibodies could significantly advance our understanding of neurodegenerative disease mechanisms, particularly in ALS research. Reports indicate that mutations within the KIFAP3 gene are associated with decreased KIFAP3 expression and increased survival in sporadic ALS, suggesting KIFAP3 as a potential therapeutic target . Researchers can employ KIFAP3 antibodies to:

• Compare KIFAP3 expression levels in various neurodegenerative conditions using brain tissue microarrays.

• Investigate the relationship between KIFAP3 and motor neuron degeneration in cell and animal models of ALS.

• Explore potential interactions between KIFAP3 and other ALS-associated proteins.

• Monitor changes in KIFAP3 expression or localization in response to potential therapeutic interventions.

What novel applications could emerge from combining KIFAP3 antibodies with advanced imaging techniques?

Integration of KIFAP3 antibodies with cutting-edge imaging technologies could unlock several innovative applications:

• Super-resolution microscopy: Visualizing KIFAP3 distribution and dynamics at nanometer resolution could reveal previously undetectable interaction patterns with kinesin motors.

• Live-cell imaging: Using membrane-permeable KIFAP3 antibody fragments could enable real-time tracking of KIFAP3 trafficking and interactions in living cells.

• Tissue clearing techniques: Combined with KIFAP3 antibodies, these approaches could enable three-dimensional visualization of KIFAP3 distribution throughout intact tissues or even whole organisms.

• Multiplex imaging: Simultaneous detection of KIFAP3 alongside multiple binding partners could map complex interaction networks, particularly relevant for understanding its role in cargo binding regulation for kinesin motors.

How can KIFAP3 antibodies facilitate studies on the role of KIFAP3 in SmgGDS-mediated signaling pathways?

KIFAP3 antibodies provide valuable tools for investigating the interaction between KIFAP3 and SmgGDS, which has important implications for small G protein regulation. Specific research strategies include:

• Co-immunoprecipitation studies using KIFAP3 antibodies to isolate and characterize KIFAP3-SmgGDS complexes across different cellular contexts.

• Proximity ligation assays to visualize and quantify KIFAP3-SmgGDS interactions at the single-molecule level within intact cells.

• Analysis of KIFAP3's role in SmgGDS-mediated regulation of small G proteins (Rho, Rap1, Ki-Ras) using KIFAP3 knockdown/overexpression approaches validated by KIFAP3 antibody detection.

• Investigation of how KIFAP3-SmgGDS interactions influence lipid-mediated post-translational modifications of small G-proteins and their subsequent membrane localization .