KIF13A Antibody

What is KIF13A and what are its key cellular functions?

KIF13A (Kinesin Family Member 13A) is a plus end-directed microtubule-dependent motor protein involved in intracellular transport processes. In humans, the canonical protein has 1805 amino acid residues with a molecular mass of 202.3 kDa . KIF13A plays critical roles in mediating the transport of mannose-6-phosphate receptor (M6PR)-containing vesicles from the trans-Golgi network to the plasma membrane through direct interaction with the AP-1 complex . Additionally, it regulates endosomal sorting during melanosome biogenesis and participates in cytokinesis . The protein is widely expressed throughout the body, with particularly high expression levels in heart, brain, and skeletal muscle tissues .

What are the key structural and localization characteristics of KIF13A?

KIF13A is predominantly localized in the Golgi apparatus and cytoplasm . The protein exists in up to five different isoforms, allowing for functional diversity across different cellular contexts . KIF13A is a member of the kinesin protein family, which consists of motor proteins that move along microtubule filaments using energy derived from ATP hydrolysis. The protein has several alternative names, including homolog of mouse KIF13A mannose-6-phosphate receptor transporter, kinesin-like protein RBKIN, and kinesin-like protein KIF13A . Evolutionarily, KIF13A is highly conserved, with orthologs identified in multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken .

What detection methods are commonly used for KIF13A in research applications?

Several immunodetection methods are routinely employed for KIF13A research. The most frequently used applications include:

For optimal immunohistochemistry results with KIF13A antibodies, antigen retrieval with TE buffer pH 9.0 is typically recommended, although citrate buffer pH 6.0 may alternatively be used . When performing Western blot analysis, HepG2 cells have been confirmed as a positive control for KIF13A expression .

How should researchers choose the appropriate KIF13A antibody for their specific experimental needs?

When selecting a KIF13A antibody, researchers should consider multiple factors beyond basic reactivity. First, determine the specific application requirements (WB, IHC, IF, IP, ELISA) as antibodies often perform differently across applications. For instance, some KIF13A antibodies are optimized for multiple applications like WB and IP, while others may be specific to a single application like ELISA .

Second, consider the species reactivity required for your experiment. Available KIF13A antibodies demonstrate varying reactivity profiles, with some specific to human samples only, and others showing cross-reactivity with mouse and rat . For cross-species studies, antibodies with documented reactivity across multiple species are essential.

Third, evaluate antibody validation data through published literature and manufacturer validation. Antibodies with citations in peer-reviewed publications provide increased confidence in performance. For example, some KIF13A antibodies have associated publications with validation figures demonstrating specificity and reproducibility .

What validation steps should be performed before using a new KIF13A antibody?

Proper validation of KIF13A antibodies is critical for reliable experimental results. A comprehensive validation process should include:

• Positive and negative controls: Use cell lines with known KIF13A expression (e.g., HepG2) as positive controls . For negative controls, consider using KIF13A knockout/knockdown samples or primary antibody omission.

• Antibody titration: Test a range of antibody dilutions to determine optimal concentration. For KIF13A antibodies, typical Western blot dilutions range from 1:500-1:1000, while IHC applications may require 1:50-1:500 dilutions .

• Specificity verification: Confirm a single band of appropriate molecular weight (202 kDa) in Western blot applications . Multiple bands might indicate degradation products, isoforms, or non-specific binding.

• Cross-reactivity assessment: If working with non-human samples, verify cross-reactivity with your species of interest, as KIF13A antibodies vary in their species reactivity profiles .

• Reproducibility testing: Perform replicate experiments to ensure consistent results across different lots and conditions.

• Subcellular localization concordance: In immunofluorescence or IHC applications, verify that the localization pattern matches expected Golgi and cytoplasmic distribution .

What are the optimal protocols for using KIF13A antibodies in Western blot applications?

For optimal Western blot detection of KIF13A, researchers should follow these methodological guidelines:

• Sample preparation: Prepare cell or tissue lysates using RIPA buffer supplemented with protease inhibitors. Given KIF13A's high molecular weight (202 kDa), complete protein extraction and denaturation are critical.

• Gel electrophoresis: Use low percentage (6-8%) SDS-PAGE gels to effectively resolve high molecular weight proteins like KIF13A. Consider gradient gels (4-15%) for better separation.

• Transfer conditions: For large proteins like KIF13A, employ wet transfer methods with extended transfer times (overnight at 30V, 4°C) to ensure complete transfer of high molecular weight proteins.

• Antibody incubation:

  • Block membranes with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Incubate with primary KIF13A antibody at 1:500-1:1000 dilution overnight at 4°C

  • Wash extensively with TBST (at least 3×10 minutes)

  • Incubate with appropriate secondary antibody (typically 1:5000-1:10000) for 1 hour at room temperature

• Detection: Use enhanced chemiluminescence detection systems with extended exposure times, as high molecular weight proteins may require longer exposures for optimal visualization.

• Controls: Include positive control samples (HepG2 cell lysate) and molecular weight markers spanning the 200 kDa range .

How can researchers optimize immunohistochemistry protocols for KIF13A detection in tissue samples?

For effective KIF13A immunohistochemical detection in tissue samples, consider these methodological recommendations:

• Tissue fixation: Use 4% paraformaldehyde or 10% neutral buffered formalin with fixation time optimized for tissue thickness (typically 24-48 hours for standard biopsies).

• Antigen retrieval: KIF13A detection specifically requires heat-induced epitope retrieval using TE buffer pH 9.0 for optimal results, though citrate buffer pH 6.0 may serve as an alternative . Perform retrieval by:

  • Heating sections in retrieval buffer to 95-100°C for 15-20 minutes

  • Allowing gradual cooling to room temperature for 20 minutes

  • Washing thoroughly with PBS before blocking

• Blocking and antibody application:

  • Block with 5-10% normal serum (matching secondary antibody host) with 0.3% Triton X-100

  • Apply KIF13A primary antibody at 1:50-1:500 dilution (optimize through titration)

  • Incubate overnight at 4°C in a humidified chamber

  • Wash extensively with PBS (3×5 minutes)

• Detection system: Use polymer-based detection systems rather than avidin-biotin methods for enhanced sensitivity and lower background when detecting KIF13A.

• Controls: Include positive control tissues (human liver cancer tissue has been validated) and negative controls (primary antibody omission and isotype controls).

What approaches can be used to study protein-protein interactions involving KIF13A?

To investigate KIF13A's protein-protein interactions, particularly its well-documented interaction with the AP-1 complex , researchers can employ multiple complementary approaches:

• Co-immunoprecipitation (Co-IP):

  • Lyse cells in non-denaturing buffer to preserve protein complexes

  • Incubate lysate with KIF13A antibody (pre-validated for IP)

  • Capture antibody-protein complexes with Protein A/G beads

  • Analyze precipitated proteins by Western blot for potential binding partners

• Proximity ligation assay (PLA):

  • This technique allows visualization of protein interactions in situ

  • Use primary antibodies against KIF13A and suspected interaction partners

  • Follow with PLA-specific secondary antibodies and amplification

  • Visualization reveals punctate signals where proteins are in close proximity (<40 nm)

• FRET/BRET analysis:

  • Create fluorescent protein fusions with KIF13A and potential partners

  • Measure energy transfer between fluorophores as indicator of protein proximity

  • Particularly useful for studying dynamic interactions in live cells

• Yeast two-hybrid screening:

  • For discovery of novel KIF13A interaction partners

  • Use KIF13A domains as bait to screen for interacting proteins

  • Validate findings with co-IP or other methods listed above

• Mass spectrometry-based interactomics:

  • Perform IP with KIF13A antibodies followed by mass spectrometry

  • Compare results to control IPs to identify specific interactors

  • Classify interactions based on abundance and specificity scores

How can KIF13A antibodies be utilized in live-cell imaging studies of intracellular transport?

Live-cell imaging of KIF13A-mediated transport requires specialized approaches that preserve protein functionality while allowing visualization:

• Antibody fragments for live-cell applications:

  • Convert conventional KIF13A antibodies into Fab fragments

  • Conjugate with cell-permeable peptides and fluorophores

  • Microinject or otherwise introduce into living cells

  • Monitor dynamics using spinning disk or lattice light-sheet microscopy

• Complementary fluorescent protein approaches:

  • Generate KIF13A-FP fusion constructs (ensuring functionality is preserved)

  • Transfect into cells of interest at low expression levels

  • Pair with labeled cargo markers (e.g., fluorescently-tagged M6PR)

  • Perform dual-color time-lapse imaging to track co-transport

• Quantitative analysis parameters:

  • Measure velocity (typically 0.5-1 μm/sec for kinesin-mediated transport)

  • Determine run length (distance traveled before dissociation)

  • Calculate pause frequency and duration

  • Assess directionality and persistence of movement

• Perturbation approaches:

  • Combine imaging with acute inhibition (e.g., photoinactivation)

  • Apply microtubule-disrupting drugs to confirm microtubule dependence

  • Use ATP depletion to verify energy requirements

  • Implement optogenetic tools for spatiotemporal control of KIF13A activity

What strategies can address challenges in detecting low-abundance KIF13A in certain tissue types?

Despite its widespread expression, KIF13A can be difficult to detect in some tissues due to low abundance. Advanced strategies to overcome this limitation include:

• Signal amplification techniques:

  • Tyramide signal amplification (TSA) can enhance immunohistochemical detection by 10-100 fold

  • For Western blot, consider enhanced chemiluminescence substrates with extended sensitivity

  • Use biotin-streptavidin amplification systems with multiple layers

• Sample enrichment approaches:

  • Perform subcellular fractionation to concentrate Golgi and cytoplasmic fractions where KIF13A localizes

  • Implement immunoprecipitation before detection to concentrate the target protein

  • Consider proximity labeling methods (BioID, APEX) to identify KIF13A-associated proteomes even when KIF13A itself is difficult to detect directly

• Alternative detection technologies:

  • Digital droplet PCR for extremely sensitive mRNA quantification

  • Single-molecule detection methods

  • Mass spectrometry with targeted multiple reaction monitoring (MRM)

• Tissue-specific optimization table:

How can researchers distinguish between different KIF13A isoforms in experimental systems?

Distinguishing between the five reported isoforms of KIF13A presents a significant challenge requiring specialized approaches:

• Isoform-specific antibody selection:

  • Carefully evaluate epitope information for available antibodies

  • Select antibodies targeting regions with isoform-specific sequences

  • Validate specificity using overexpression systems with individual isoforms

• PCR-based isoform detection:

  • Design primer pairs spanning exon junctions specific to each isoform

  • Perform RT-PCR or qPCR with isoform-specific primers

  • Validate amplicon identity through sequencing

  • Consider digital PCR for absolute quantification of isoform ratios

• Mass spectrometry approaches:

  • Analyze tryptic digests for isoform-specific peptides

  • Implement parallel reaction monitoring for targeted detection

  • Quantify isoform-specific peptides for relative abundance determination

• Functional differentiation strategies:

  • Generate isoform-specific knockdowns using siRNAs targeting unique regions

  • Perform rescue experiments with individual isoforms

  • Assess functional readouts (e.g., M6PR transport efficiency, melanosome distribution)

• Combined detection workflow:

  • Initial screening with pan-KIF13A antibodies by Western blot

  • High-resolution gel electrophoresis to separate closely related isoforms

  • Confirmation using isoform-specific detection methods

  • Correlation with functional assays to determine isoform-specific roles

How can researchers address non-specific binding issues when using KIF13A antibodies?

Non-specific binding presents a common challenge when working with KIF13A antibodies. Systematic troubleshooting approaches include:

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time (2-3 hours or overnight)

  • Include detergents (0.1-0.3% Triton X-100 or Tween-20) to reduce hydrophobic interactions

  • Consider adding 1-5% non-fat dry milk to both blocking and antibody diluent solutions

• Antibody dilution optimization:

  • Perform sequential dilution series to identify optimal concentration

  • For Western blot, test range from 1:250 to 1:2000

  • For IHC applications, evaluate dilutions from 1:25 to 1:500

  • Remember that more concentrated antibody does not always yield better results

• Washing protocol enhancement:

  • Increase number of washes (minimum 5-6 washes)

  • Extend wash duration (15-20 minutes per wash)

  • Use gentle agitation during washing

  • Consider adding 0.5M NaCl to wash buffer to disrupt low-affinity interactions

• Pre-adsorption controls:

  • Pre-incubate antibody with immunizing peptide (if available)

  • Compare staining patterns with and without pre-adsorption

  • Specific signals should disappear after pre-adsorption

• Validation across multiple detection methods:

  • Confirm findings using at least two independent detection techniques

  • Compare patterns between fresh and fixed samples where possible

  • Correlate protein detection with mRNA expression data

What strategies can resolve issues with detecting the full-length KIF13A protein (202 kDa) in Western blot applications?

The high molecular weight of KIF13A (202 kDa) presents specific technical challenges in Western blot applications. To effectively detect the full-length protein:

• Sample preparation optimization:

  • Use strong lysis buffers (RIPA with 0.5% sodium deoxycholate)

  • Add protease inhibitor cocktails immediately after lysis

  • Minimize freeze-thaw cycles to prevent degradation

  • Consider adding phosphatase inhibitors to preserve post-translational modifications

  • Sonicate briefly to shear DNA without damaging proteins

• Gel electrophoresis adjustments:

  • Use gradient gels (4-15%) or low percentage (6%) gels

  • Reduce voltage during electrophoresis (60-80V)

  • Extend running time to improve resolution of high molecular weight proteins

  • Include molecular weight markers that extend beyond 200 kDa

  • Pre-chill running buffer to minimize heat-induced protein degradation

• Transfer optimization for high-molecular-weight proteins:

  • Implement wet transfer systems rather than semi-dry

  • Add 0.05-0.1% SDS to transfer buffer to improve elution of large proteins

  • Reduce methanol concentration to 10% to prevent gel hardening

  • Extend transfer time (overnight at 30V, 4°C)

  • Verify transfer efficiency using reversible staining of membranes

• Detection system considerations:

  • Select high-sensitivity chemiluminescent substrates

  • Extend exposure times (may require 5-15 minutes versus standard 1-5 minutes)

  • Consider fluorescent Western blot systems for better linear dynamic range

  • Use fresh antibody dilutions to maximize binding efficiency

• Positive controls and validation:

  • Include HepG2 cell lysates as positive controls for KIF13A expression

  • Consider running recombinant KIF13A (full-length or domain) as size reference

  • Verify specificity through knockdown experiments when possible

How can researchers differentiate between true KIF13A signal and potential cross-reactivity with related kinesin family members?

The kinesin superfamily contains multiple related proteins with structural similarities that can lead to antibody cross-reactivity. To ensure KIF13A-specific detection:

• Antibody selection criteria:

  • Choose antibodies raised against unique regions of KIF13A rather than conserved motor domains

  • Evaluate manufacturer cross-reactivity testing data

  • Select antibodies validated in knockout/knockdown systems

  • Consider monoclonal antibodies for increased specificity

• Validation through genetic approaches:

  • Perform siRNA/shRNA knockdown of KIF13A and verify signal reduction

  • Implement CRISPR/Cas9 knockout systems as definitive controls

  • Conduct overexpression studies with tagged KIF13A constructs

  • Compare detection patterns between these systems

• Bioinformatic cross-reactivity analysis:

  • Compare epitope sequences against related kinesin family members

  • Predict potential cross-reactive proteins through sequence homology

  • Design experiments to specifically rule out detected cross-reactivity

• Comparative detection strategies:

  • Use multiple antibodies targeting different KIF13A epitopes

  • Compare detection patterns for consistency

  • Implement peptide competition assays with KIF13A-specific peptides

  • Include parallel detection of closely related kinesins (e.g., KIF13B) for comparison

• Functional validation approaches:

  • Correlate protein detection with known KIF13A functions (e.g., M6PR trafficking)

  • Assess subcellular localization consistency with expected Golgi/cytoplasmic distribution

  • Verify interaction with known binding partners through co-IP experiments

How can KIF13A antibodies be utilized in studying neurodegenerative diseases?

Given KIF13A's high expression in brain tissue and its crucial role in intracellular transport, it presents a significant target for neurodegenerative disease research:

• Pathological sample analysis:

  • Compare KIF13A expression and localization patterns between healthy and diseased brain tissues

  • Evaluate KIF13A function in various neurodegenerative conditions including Alzheimer's, Parkinson's, and ALS

  • Assess potential co-localization with disease-specific aggregates or markers

  • Determine if KIF13A transport is impaired in conditions with known axonal transport defects

• Mechanistic investigation approaches:

  • Examine KIF13A-mediated transport of specific neuronal cargoes

  • Assess potential interactions between KIF13A and disease-associated proteins

  • Evaluate the impact of disease-relevant stressors on KIF13A function

  • Implement live-cell imaging in primary neurons to visualize transport dynamics

• Therapeutic targeting strategies:

  • Screen for compounds that modulate KIF13A activity or expression

  • Evaluate the potential of KIF13A as a biomarker for disease progression

  • Assess whether restoration of compromised KIF13A function improves neuronal health

  • Develop targeted delivery systems utilizing KIF13A transport mechanisms

• Model system applications:

  • Implement KIF13A antibodies in iPSC-derived neurons from patients

  • Utilize animal models with KIF13A mutations or manipulations

  • Correlate findings across species to establish evolutionary conservation of mechanisms

  • Develop high-throughput screening systems for KIF13A functional assessment

What are the current challenges and solutions in studying post-translational modifications of KIF13A?

Post-translational modifications (PTMs) likely play critical roles in regulating KIF13A function, but studying these modifications presents unique challenges:

• PTM-specific detection approaches:

  • Develop and validate phospho-specific antibodies for key regulatory sites

  • Implement enrichment strategies (e.g., phosphopeptide enrichment) before analysis

  • Use mass spectrometry-based approaches for unbiased PTM mapping

  • Apply proximity labeling methods to identify enzymes regulating KIF13A modifications

• Functional correlation strategies:

  • Generate phosphomimetic and phospho-deficient mutants of key sites

  • Assess impact on KIF13A localization, motor activity, and cargo binding

  • Implement live-cell imaging with biosensors to monitor dynamic modifications

  • Correlate modifications with specific cellular states or stimuli

• Technical challenges and solutions table:

• Integrative analysis workflow:

  • Initial PTM identification through mass spectrometry

  • Validation with site-specific antibodies when available

  • Functional characterization through mutagenesis

  • Correlation with cellular contexts and stimuli

  • Computational modeling of modification impacts on protein structure and function

How can multiplexed imaging approaches be optimized for studying KIF13A in complex tissue environments?

Advanced multiplexed imaging allows simultaneous visualization of KIF13A alongside multiple markers to understand its contextual relationships in tissues:

• Multiplexed immunofluorescence strategies:

  • Sequential immunostaining with antibody stripping between rounds

  • Spectral unmixing approaches for simultaneous multi-color imaging

  • Implement tyramide signal amplification for detecting low-abundance targets

  • Use DNA-barcoded antibodies for highly multiplexed detection (CODEX, Imaging Mass Cytometry)

• Antibody panel design considerations:

  • Include KIF13A antibodies validated for tissue applications

  • Combine with markers for subcellular compartments (Golgi, endosomes, plasma membrane)

  • Add antibodies for known interaction partners (AP-1 complex components)

  • Include cell-type specific markers for contextual analysis

• Image analysis pipeline optimization:

  • Implement machine learning approaches for signal segmentation

  • Develop quantitative colocalization metrics

  • Use spatial statistics to analyze distribution patterns

  • Apply trajectory analysis for transport studies in tissue contexts

• Validation controls for multiplexed approaches:

  • Single-stain controls for spectral unmixing

  • Isotype controls matched to each primary antibody

  • Absorption controls to verify signal specificity

  • Replication across multiple samples and conditions

• Advanced tissue preparation considerations:

  • Optimize fixation to preserve KIF13A epitopes while enabling multiplexed detection

  • Consider clearing techniques (CLARITY, iDISCO) for thick tissue sections

  • Implement expanded microscopy for improved resolution of transport complexes

  • Use precision-cut tissue slices for live imaging applications in near-native environments