KIFAP3 Antibody, FITC conjugated

What is KIFAP3 and why is it significant in research?

KIFAP3 (Kinesin Associated Protein 3) is a protein that functions as part of the kinesin motor complex involved in intracellular transport processes. Its significance extends beyond structural functions, as recent research has implicated KIFAP3 in transcriptional regulation. KIFAP3 pre-mRNA accumulation patterns have been observed to change during transcription processes, with studies showing that RPRD2 depletion can accelerate KIFAP3 transcription by approximately 10 minutes compared to control samples . This connection to transcriptional control mechanisms makes KIFAP3 a protein of interest in fundamental cellular biology research and disease studies, particularly amyotrophic lateral sclerosis (ALS).

What epitopes are targeted by commercially available KIFAP3 antibodies?

Available KIFAP3 antibodies target multiple epitopes across the protein sequence. Current research-grade antibodies include those targeting amino acids 660-792, 1-200, N-terminal regions, middle regions (683-732), and other specific domains . This diversity allows researchers to select antibodies that target regions of interest for their specific experimental questions. For instance, antibodies targeting the N-terminal region may be preferable for studying protein-protein interactions, while those targeting other domains might be more suitable for detecting specific isoforms or post-translational modifications.

What detection methods are compatible with KIFAP3 antibodies?

KIFAP3 antibodies have demonstrated compatibility with multiple experimental methodologies including Western blotting (WB), immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and immunofluorescence (IF) . Each application requires specific optimization parameters, with FITC-conjugated variants being particularly valuable for direct fluorescence detection in IF and flow cytometry applications without requiring secondary antibody incubation steps.

How should experimental controls be designed when using KIFAP3 antibodies in gene knockdown studies?

When using KIFAP3 antibodies in knockdown experiments, comprehensive control strategies are essential. Researchers should implement: (1) non-targeting siRNA/shRNA controls, (2) multiple independent targeting sequences to rule out off-target effects, (3) rescue experiments with exogenous expression of siRNA-resistant KIFAP3, and (4) demonstration of knockdown efficiency at both RNA and protein levels. In studies examining KIFAP3's role in transcription, researchers have successfully employed RNA metabolic labeling with 4sU following KIFAP3-associated protein knockdowns, comparing nascent RNA synthesis patterns to suitable controls . This methodology can detect subtle changes in transcription rates that might be missed by steady-state RNA measurements.

What are optimal fixation and permeabilization protocols for FITC-conjugated KIFAP3 antibodies in immunofluorescence?

The optimal fixation protocol for FITC-conjugated KIFAP3 antibodies involves a balanced approach that preserves both epitope accessibility and fluorophore activity. For adherent cell lines, 4% paraformaldehyde fixation (10 minutes at room temperature) followed by gentle permeabilization with 0.1% Triton X-100 (5 minutes) generally yields favorable results. Critical parameters include: (1) avoiding over-fixation which can mask epitopes, (2) using phosphate buffers rather than Tris-based buffers during fixation to prevent FITC quenching, (3) including 0.1% BSA during antibody incubation to reduce non-specific binding, and (4) minimizing exposure to light throughout the protocol to prevent photobleaching of the FITC conjugate.

How can researchers determine optimal antibody dilutions for specific KIFAP3 detection methods?

Determining optimal antibody dilutions requires systematic titration experiments. For the FITC-conjugated anti-KIFAP3 antibody targeting amino acids 660-792, researchers should perform parallel experiments using 2-fold serial dilutions (typically starting from 1:50 to 1:800) and evaluate signal-to-noise ratios across applications. A quantitative approach involves plotting signal intensity against antibody concentration to identify the inflection point where signal plateaus relative to background. For immunofluorescence applications with FITC-conjugated antibodies, special consideration should be given to autofluorescence controls and single-stain controls when performing multiplex experiments.

How should researchers interpret contradictory findings regarding KIFAP3's association with ALS survival?

When interpreting such contradictions, researchers should consider: (1) differences in cohort composition (specialty clinic patients versus population-based samples), (2) sample size limitations in expression studies (26 samples in the original study versus 144 samples in the replication), (3) methodological differences in expression quantification, and (4) population-specific genetic factors. The study notes that longer-living patients are more likely to attend specialty clinics and access treatments that extend survival, potentially introducing selection bias . These contradictions highlight the importance of validating findings across multiple independent cohorts with careful attention to cohort composition.

What causes variability in KIFAP3 antibody performance across different tissues and applications?

Variability in KIFAP3 antibody performance can be attributed to multiple factors that researchers must consider when interpreting results. First, epitope accessibility varies between applications—proteins denatured for Western blot may expose epitopes hidden in native conformation techniques like immunoprecipitation. Second, post-translational modifications may obscure epitopes in tissue-specific patterns. Third, cross-reactivity profiles differ between antibodies; the polyclonal antibody targeting amino acids 660-792 shows reactivity with human samples, while others demonstrate cross-reactivity with multiple species including mouse, rat, and other mammals .

When confronted with variable performance, researchers should implement a multi-antibody approach. For instance, studies investigating KIFAP3 protein levels have employed two distinct antibodies (Santa Cruz Biotechnology sc-55598 and BD Biosciences 610637) in parallel Western blot experiments to validate findings . This strategy provides robustness against antibody-specific artifacts.

What spectral considerations are important when using FITC-conjugated KIFAP3 antibodies in multiplex imaging?

When designing multiplex imaging experiments with FITC-conjugated KIFAP3 antibodies, researchers must account for FITC's spectral properties: excitation maximum at approximately 495nm and emission maximum around 519nm. This creates practical considerations: (1) avoid fluorophores with significant spectral overlap like GFP (similar emission spectrum) or TRITC (potential FRET interactions); (2) implement appropriate compensation controls when using flow cytometry; (3) select complementary fluorophores like Cy5 (649/670nm) that provide clean spectral separation; and (4) consider sequential imaging approaches when using confocal microscopy to eliminate bleed-through artifacts.

The linear unmixing capabilities of modern confocal systems can help separate overlapping signals, but this requires careful single-fluorophore controls. Additionally, researchers should be aware that FITC is susceptible to photobleaching and pH sensitivity (fluorescence decreases below pH 7.0), requiring appropriate buffers and antifade reagents during sample preparation and imaging.

How can researchers validate the specificity of FITC-conjugated KIFAP3 antibodies?

Validating antibody specificity is crucial for reliable research. For FITC-conjugated KIFAP3 antibodies, researchers should implement a multi-faceted approach: (1) genetic validation using KIFAP3 knockdown or knockout models to demonstrate signal reduction; (2) peptide competition assays using the immunogen peptide (amino acids 660-792) to confirm epitope-specific binding; (3) comparison with non-conjugated primary antibodies against the same epitope followed by FITC-conjugated secondary antibodies to confirm similar staining patterns; and (4) Western blot validation showing a band of appropriate molecular weight (approximately 110-120 kDa for full-length KIFAP3).

Additionally, researchers can validate KIFAP3 antibodies through immunoprecipitation followed by mass spectrometry to confirm the identity of the precipitated protein. Studies investigating KIFAP3 expression have employed multiple detection methods including Taqman gene expression assays (hs00183973 and hs00946074) and protein detection via Western blotting to ensure robust findings .

How should researchers design experiments to evaluate KIFAP3's role in transcriptional regulation?

Designing experiments to investigate KIFAP3's role in transcription requires a multifaceted approach targeting both expression dynamics and functional outcomes. RNA metabolic labeling with 4sU followed by sequencing of isolated labeled RNA has proven effective for measuring nascent RNA synthesis following manipulation of KIFAP3-associated proteins . This approach allows detection of rapid changes in transcription before steady-state levels are affected.

A comprehensive experimental design should include: (1) genetically manipulating KIFAP3 expression using siRNA/shRNA knockdown or CRISPR-based approaches; (2) performing RNA-seq on both nascent transcripts (via metabolic labeling) and steady-state RNA; (3) conducting chromatin immunoprecipitation (ChIP) to examine KIFAP3 association with chromatin and potential co-localization with RNA polymerase II; and (4) implementing transcription elongation rate assays using DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) retraction protocols. The latter approach has revealed that depletion of RPRD2, a protein that interacts with the transcriptional machinery, accelerates KIFAP3 transcription by approximately 10 minutes compared to control conditions .

What contradictions exist regarding KIFAP3's role in ALS pathogenesis?

Furthermore, the proposed mechanism linking rs1541160 genotype to KIFAP3 expression has been challenged. The original finding that the CC genotype reduced expression was based on 26 brain samples, while later studies using 144 samples from four brain regions found no correlation between the SNP and expression levels . Additional analyses using Taqman gene expression assays and semiquantitative Western blotting with two different antibodies also failed to detect genotype-dependent differences in KIFAP3 expression .

These contradictions highlight important considerations for ALS genetics research: (1) the potential impact of cohort composition and selection bias on genetic association findings; (2) the importance of large sample sizes for expression quantitative trait loci (eQTL) studies; and (3) the need for functional validation of proposed disease-modifying mechanisms.

What strategies can address non-specific background when using FITC-conjugated KIFAP3 antibodies?

Non-specific background is a common challenge with FITC-conjugated antibodies due to tissue autofluorescence in the green spectrum and potential Fc-receptor interactions. Effective mitigation strategies include: (1) implementing a dual blocking approach with both 5% normal serum from the same species as the samples and 1% BSA; (2) including 0.1-0.3% Triton X-100 in blocking solutions to reduce hydrophobic interactions; (3) performing longer wash steps (4-5 washes of 5 minutes each) with 0.1% Tween-20 in PBS; and (4) utilizing Sudan Black B (0.1% in 70% ethanol) post-staining to quench lipofuscin-based autofluorescence in tissues with high lipid content.

For particularly challenging samples, researchers can employ antibody dilution optimization experiments comparing signal-to-noise ratios across different antibody concentrations and incubation conditions. Additionally, pre-adsorption of the antibody with tissue lysates from species with expected cross-reactivity but lacking the target protein can reduce non-specific binding.

How can researchers incorporate KIFAP3 detection into quantitative cellular analyses?

Quantitative analysis of KIFAP3 localization and expression requires rigorous image acquisition and analysis protocols. For FITC-conjugated antibodies, researchers should: (1) establish consistent exposure settings based on samples with the highest expected signal; (2) implement flat-field correction to account for illumination heterogeneity; (3) develop automated segmentation protocols that identify subcellular compartments of interest; and (4) extract multiple parameters beyond mean intensity, including integrated density, coefficient of variation, and correlation with other markers in multiplex experiments.

Advanced applications include high-content screening approaches where KIFAP3 localization changes are quantified across treatment conditions or genetic perturbations. When coupled with machine learning classification algorithms, these approaches can identify subtle phenotypes not apparent through visual inspection alone. For transcription studies, quantitative co-localization analysis of KIFAP3 with transcriptional machinery components can provide insights into functional interactions during various phases of transcription.