KIF25 Antibody, Biotin conjugated

What is KIF25 and why is it significant in cellular research?

KIF25 (Kinesin Family Member 25) is a minus-end microtubule-dependent motor protein that acts as a negative regulator of centrosome separation required to prevent premature centrosome separation during interphase. It's essential for maintaining a centered nucleus to ensure stable spindle orientation at the onset of mitosis . KIF25 may also function as a negative regulator of amino acid starvation-induced autophagy . The significance of KIF25 in research stems from its involvement in critical cellular processes related to cell division and transport mechanisms, making it a valuable target for studying cytoskeletal dynamics and associated pathologies.

What is the principle behind biotin conjugation in antibodies and how does it enhance detection?

Biotin conjugation exploits one of the strongest non-covalent interactions in nature - the binding between biotin and (strept)avidin proteins with dissociation constants (KD) of 10^-14-10^-15, which is 10^3-10^6 times higher than typical antigen-antibody interactions . This system allows for:

The biotin's small size (240 Da) and flexible valeric side chain enable conjugation to antibodies without disrupting their binding properties . When a biotinylated antibody binds to its target, the biotin moieties become available to bind streptavidin conjugated with detection molecules (enzymes, fluorophores), creating a powerful signal amplification system particularly valuable for detecting low-abundance targets .

How do KIF25 antibodies differ from other kinesin family member antibodies in research applications?

KIF25 antibodies target a unique kinesin protein that lacks ATPase activity but remains constitutively associated with microtubules . Unlike other kinesin family members like KIF26B (involved in embryonic kidney development) or the KIF5 family (involved in GABAAR transport) , KIF25 functions primarily in centrosome dynamics and nuclear positioning. This specificity makes KIF25 antibodies particularly valuable for studying:

• Mitotic spindle organization

• Nuclear positioning mechanisms

• Centrosome separation regulation

• Potential roles in autophagy regulation

When selecting between different kinesin antibodies, researchers should consider the specific cellular processes and organelles they aim to investigate.

What are the optimal protocols for using KIF25 antibody, biotin conjugated in immunohistochemistry?

For optimal immunohistochemistry (IHC) with biotin-conjugated KIF25 antibodies:

• Sample preparation: Fix tissues with 4% paraformaldehyde or 10% neutral buffered formalin, followed by paraffin embedding and sectioning at 4-6 μm thickness.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0) for 15-20 minutes.

• Blocking: Block endogenous biotin using a biotin blocking kit to prevent non-specific binding, especially important when using biotin-conjugated primary antibodies .

• Antibody dilution: Use at 1:150 dilution for paraffin sections , though optimization may be required for specific applications.

• Detection: Apply streptavidin conjugated with enzyme (HRP) or fluorophore, following manufacturer's recommendations. Streptavidin can bind up to four biotin molecules, allowing signal amplification .

• Controls: Include both positive controls (tissues known to express KIF25) and negative controls (omission of primary antibody) to validate specificity.

Using a spacer-extended biotin conjugate (Biotin-SP) can improve sensitivity by extending the biotin moiety away from the antibody surface, making it more accessible to streptavidin binding sites .

How can I optimize biotin-conjugated KIF25 antibody concentration for Western blot applications?

Optimization of biotin-conjugated KIF25 antibody for Western blot requires a systematic approach:

• Initial titration: Begin with a range of dilutions (1:500, 1:1000, 1:2000) as recommended by manufacturers .

• Protein loading: Load 20-50 μg of total protein per lane, with higher amounts for detecting low-abundance proteins.

• Blocking optimization: Use 5% BSA in TBST to block membranes, as milk contains biotin that may interfere with streptavidin binding.

• Streptavidin detection system: Use streptavidin-HRP at optimized concentrations (typically 1:5000-1:20000) for detection.

• Exposure time optimization: Begin with short exposures (30 seconds) and increase incrementally to avoid signal saturation while capturing specific bands.

• Analysis: Evaluate signal-to-noise ratio and band specificity to determine optimal antibody concentration.

The expected molecular weight of human KIF25 is approximately 35.1 kDa , which serves as a reference point for validating specific binding.

What methods are available for determining the biotin-to-protein ratio in KIF25 antibody preparations?

Determining the biotin-to-protein (B/P) ratio is critical for quality control of biotinylated antibodies. Several methods with varying sensitivity and reproducibility are available:

• Modified Quant*Tag method: Offers superior reproducibility with %CV not exceeding 4.2% at various challenge ratios. This method can reliably measure small-scale biotinylation (≥0.1 nmol/well in a 384-well plate) with low B/P ratios (<5) .

• HABA assay: Widely used but has insufficient sensitivity and reproducibility for rigorous quality control. The method measures the displacement of HABA from avidin by biotin .

• FluoReporter® Biotin Quantitation Kit: Provides another approach with different sensitivity and dynamic range compared to HABA and Quant*Tag methods .

• Capillary isoelectric focusing: Can detect as little as 10% unconjugated protein in biotin-antibody preparations, allowing assessment of conjugation efficiency .

For KIF25 antibodies, challenge ratios (CR) of biotin to protein between 5-20 are typically used, with the median percentage incorporation across various antibodies being approximately 46% .

How can I address biotin interference issues in immunoassays using KIF25 biotin-conjugated antibodies?

Biotin interference is a significant concern in immunoassays using biotin-conjugated antibodies, with reports of false results due to high biotin levels in samples . To address this:

• Sample pretreatment: Remove excess biotin from samples using streptavidin-coated magnetic beads or specialized removal kits.

• Alternative detection systems: Consider using non-biotin detection systems when analyzing samples from subjects taking biotin supplements.

• Dilution protocols: Implement sample dilution protocols to reduce biotin concentrations below interference thresholds.

• Timing considerations: If possible, collect samples at least 8 hours after the last biotin dose when working with clinical samples.

• Blocking strategies: Use specialized blocking reagents designed to prevent endogenous biotin interference.

• Validation: Include biotin spike-in controls to assess potential interference levels in your specific assay format.

The FDA has issued safety alerts regarding biotin interference in diagnostic tests , highlighting the importance of addressing this issue in research applications as well.

What quality control parameters should be monitored when using biotin-conjugated KIF25 antibodies?

Critical quality control parameters include:

• Biotin-to-protein ratio: Optimal B/P ratios typically range from 3-7 for antibodies . Either under- or over-biotinylation can compromise performance.

• Presence of unconjugated antibody: Residual unconjugated antibody acts as interference that suppresses assay signals. Capillary isoelectric focusing can detect as little as 10% unconjugated material .

• Specificity verification: Confirm target specificity through appropriate controls, particularly important as conjugation might affect binding characteristics.

• Storage integrity: Monitor for activity loss during storage, as biotin-conjugated antibodies may have different stability profiles than unconjugated versions.

• Batch consistency: Assess lot-to-lot variability as biotin incorporation can vary from 30% to 70% between different antibodies even under identical conjugation conditions .

• Functionality testing: Verify that conjugation hasn't impaired antibody function using application-specific positive controls.

Manufacturers typically purify KIF25 biotin-conjugated antibodies to >95% purity using protein G or affinity chromatography methods .

What are the best storage conditions to maintain stability of biotin-conjugated KIF25 antibodies?

To maintain optimal stability and activity of biotin-conjugated KIF25 antibodies:

• Temperature: Store at -20°C or -80°C for long-term storage . Avoid repeated freeze-thaw cycles.

• Formulation: Antibodies are typically provided in stabilizing buffers containing:

  • 50% Glycerol

  • PBS (pH 7.4)

  • 0.01-0.03% preservative (e.g., Proclin 300 or sodium azide)

  • Sometimes 0.25% BSA for added stability

• Aliquoting: Upon receipt, divide into small working aliquots to avoid repeated freeze-thaw cycles.

• Short-term storage: For daily use, store at 4°C in the dark for up to two weeks .

• Shipping conditions: Typically shipped with blue ice (4°C) , which maintains antibody integrity during transit.

• Handling: When removing from storage, thaw completely but gently at room temperature or 4°C, avoid heat, and mix thoroughly before use.

Following these guidelines will help maintain reactivity and specificity throughout the antibody's shelf life.

How can biotinylated KIF25 antibodies be utilized in multi-labeling immunofluorescence experiments?

Biotinylated KIF25 antibodies offer significant advantages in multi-labeling experiments:

• Sequential labeling strategy: Apply biotinylated KIF25 antibody first, followed by streptavidin-conjugated fluorophore (e.g., Cy3, Alexa Fluor 488), then block remaining biotin/streptavidin binding sites before applying additional primary-secondary antibody pairs with different fluorophores.

• Signal amplification: Utilize branched streptavidin systems where biotinylated KIF25 antibodies bind streptavidin, which can then bind additional biotinylated detection molecules, creating an amplified signal for low-abundance targets .

• Quantum dot labeling: Exploit the exceptional photostability of quantum dot-neutravidin conjugates with biotinylated KIF25 antibodies for long-term observation of trafficking during live imaging .

• Combining with FRET techniques: Implement Förster resonance energy transfer by using biotinylated KIF25 antibodies with donor fluorophore-conjugated streptavidin alongside acceptor fluorophore-labeled antibodies against interacting proteins .

• Integration with super-resolution microscopy: The high specificity of the biotin-streptavidin system makes it suitable for techniques requiring precise localization like STORM or PALM.

These approaches enable simultaneous visualization of KIF25 alongside other cellular components to study its interactions and localization patterns.

What is known about KIF25's role in microtubule dynamics and how can biotinylated antibodies help elucidate this function?

KIF25's role in microtubule dynamics remains partially characterized, but current research indicates it:

• Functions as a minus-end microtubule-dependent motor protein

• Acts as a negative regulator of centrosome separation during interphase

• Maintains nuclear positioning to ensure stable spindle orientation

• May regulate amino acid starvation-induced autophagy

Biotinylated KIF25 antibodies can help elucidate these functions through:

• Live cell imaging: Combining biotinylated KIF25 antibodies with cell-permeable streptavidin-fluorophore conjugates to track KIF25 dynamics in real-time.

• Proximity labeling: Using biotinylated KIF25 antibodies with streptavidin-conjugated peroxidases for APEX/BioID approaches to identify proximal proteins.

• Pull-down assays: Leveraging the strong biotin-streptavidin interaction for efficient and specific immunoprecipitation of KIF25 and associated proteins.

• Electron microscopy studies: Using gold-toned silver-intensified nanogold particles conjugated to streptavidin to visualize precise subcellular localization of KIF25 relative to microtubule structures, similar to approaches used with KIF5 .

• Functional inhibition studies: Delivering blocking antibodies to disrupt KIF25 function and observe effects on centrosome dynamics and nuclear positioning.

These approaches could help determine whether KIF25, like other kinesin family members, forms motor protein complexes for cargo delivery or has distinct regulatory functions in microtubule organization.

How can biotinylated KIF25 antibodies be integrated into high-throughput screening platforms?

Integration of biotinylated KIF25 antibodies into high-throughput screening platforms can accelerate discovery in several research areas:

• Microarray-based screening: Immobilize biotinylated KIF25 antibodies on streptavidin-coated microarray surfaces to capture KIF25 and associated complexes from cell lysates, enabling rapid screening of:

  • Protein-protein interactions

  • Post-translational modifications

  • Effects of small molecule inhibitors on complex formation

• Automated immunocytochemistry: Implement in cell-based assays using high-content imaging systems to screen:

  • Compounds affecting KIF25 localization

  • siRNA libraries for genes influencing KIF25 function

  • CRISPR libraries for genetic modifiers of KIF25 activity

• Flow cytometry applications: Use biotinylated KIF25 antibodies with streptavidin-fluorophore conjugates in high-throughput flow cytometry to:

  • Quantify KIF25 expression levels across cell populations

  • Correlate KIF25 levels with cell cycle phases

  • Assess effects of treatments on KIF25 expression

• Bead-based multiplex assays: Couple biotinylated KIF25 antibodies to distinct streptavidin-coated bead populations in multiplex bead arrays to simultaneously analyze:

  • KIF25 protein levels

  • Associated proteins

  • Phosphorylation states

This integration enables rapid screening of thousands of conditions, accelerating the discovery of KIF25 functions, regulatory mechanisms, and potential therapeutic targets.

What are the methodological considerations when using biotinylated KIF25 antibodies for studying protein-protein interactions?

When using biotinylated KIF25 antibodies to study protein-protein interactions:

• Cross-linking strategies: Consider using cleavable cross-linkers before immunoprecipitation to stabilize transient interactions. Optimization is crucial as excessive cross-linking may mask epitopes recognized by the antibody.

• Competition controls: Include excess non-biotinylated KIF25 antibody to demonstrate specificity of captured interactions.

• Wash stringency optimization: Balance between preserving weak interactions and removing non-specific binding. A stepwise washing protocol with decreasing stringency buffers may help identify interaction strength profiles.

• Buffer composition: Optimize salt concentration, detergent type/concentration, and pH to maintain native protein interactions while allowing efficient antibody binding.

• Mass spectrometry compatibility: When planning downstream MS analysis, consider:

  • Using MS-compatible cross-linkers

  • Implementing on-bead digestion protocols

  • Including isotopically labeled reference proteins for quantification

• Validation approaches: Confirm identified interactions through reciprocal immunoprecipitation, proximity ligation assays, or FRET techniques.

• Subcellular fractionation: Consider fractionating samples before immunoprecipitation to enrich for interactions occurring in specific cellular compartments.

Similar approaches have successfully identified interactions between kinesin family proteins and adaptors, such as the HAP1-KIF5 complex involved in GABAAR trafficking .

How can biotinylated KIF25 antibodies be utilized for studying disease mechanisms?

Biotinylated KIF25 antibodies offer powerful tools for investigating disease mechanisms through several approaches:

• Neurodegenerative disease research: Given the importance of kinesin motor proteins in axonal transport, biotinylated KIF25 antibodies can be used in models of diseases like Huntington's, similar to studies with KIF5 that revealed disrupted GABAAR trafficking due to mutant huntingtin .

• Cancer biomarker studies: Implement biotinylated KIF25 antibodies in multiplex biomarker detection platforms to assess correlation between KIF25 expression/localization and cancer progression or treatment response.

• Tissue microarray analysis: Apply to patient tissue microarrays to evaluate KIF25 expression patterns across disease stages and correlate with clinical outcomes.

• Therapeutic antibody development: Use as a starting point for developing antibody-drug conjugates (ADCs) through biotin-streptavidin linking systems, similar to approaches using streptavidin-linked antibodies conjugated to biotinylated toxins .

• In vivo imaging: Develop biotinylated KIF25 antibody fragments compatible with in vivo administration coupled with streptavidin-conjugated imaging agents for visualizing KIF25 expression in disease models.

• Extracellular vesicle (EV) studies: Employ biotinylated KIF25 antibodies to capture and characterize EVs potentially carrying KIF25 as cargo in disease states.

These applications could help establish whether alterations in KIF25 function contribute to pathological processes, similar to how disrupted kinesin-dependent transport has been implicated in neurodegeneration .

What are the considerations for using biotinylated KIF25 antibodies in combination with super-resolution microscopy techniques?

Implementing biotinylated KIF25 antibodies in super-resolution microscopy requires specific considerations:

• Labeling density optimization: The distance between fluorophores significantly impacts resolution. Titrate both biotinylated KIF25 antibody and streptavidin-fluorophore concentrations to achieve optimal labeling density for techniques like STORM or PALM.

• Fluorophore selection: Choose streptavidin conjugated with fluorophores specifically optimized for super-resolution techniques:

  • For STORM: Alexa Fluor 647 or Cy5 conjugates

  • For STED: STAR 580 or STAR 635P conjugates

  • For PALM: Photoactivatable fluorescent protein fusions to streptavidin

• Sample preparation protocols: Implement specialized fixation methods that preserve nanoscale structures while maintaining antibody accessibility:

  • Use extraction before fixation to reduce cytoplasmic background

  • Consider specialized embedding media with matching refractive indices

  • Apply drift correction beads for long acquisition protocols

• Validation approaches: Confirm super-resolution findings using complementary techniques:

  • Correlative light and electron microscopy

  • Proximity ligation assays

  • FRET-based approaches

• Signal amplification considerations: While the biotin-streptavidin system offers amplification, excessive amplification steps can increase the apparent size of structures beyond actual dimensions.

• Multicolor imaging strategies: For co-localization studies, carefully select fluorophore combinations with minimal spectral overlap or implement sequential imaging protocols.

These approaches could reveal previously unrecognized details of KIF25 localization and its association with microtubules and other cellular structures.

What novel drug delivery applications might be possible using biotinylated KIF25 antibodies?

Biotinylated KIF25 antibodies could enable several innovative drug delivery approaches:

• Nanoparticle targeting: Functionalize nanoparticle surfaces with streptavidin for binding biotinylated KIF25 antibodies, creating a versatile platform for delivering:

  • Small molecule inhibitors

  • siRNA/mRNA therapeutics

  • Protein therapeutics

  This approach has been successfully applied with other antibodies for targeting specific cell populations .

• Antibody-drug conjugates (ADCs): Utilize the biotin-streptavidin system to rapidly evaluate potential KIF25-targeted ADCs without extensive chemical conjugation, similar to approaches that conjugate streptavidin-linked antibodies to biotinylated payloads .

• Blood-brain barrier (BBB) targeting: Combine with molecular Trojan horse technology by creating fusion proteins between BBB-crossing antibodies (e.g., transferrin receptor antibodies) and streptavidin for binding biotinylated KIF25 antibodies, enabling CNS delivery .

• Intracellular delivery systems: Develop cell-penetrating peptide-streptavidin conjugates that can bind biotinylated KIF25 antibodies for intracellular targeting of specific KIF25-associated processes.

• Theranostic applications: Create dual-function platforms where biotinylated KIF25 antibodies bound to streptavidin nanoparticles carry both imaging agents and therapeutic payloads.

These applications could be particularly valuable if KIF25 expression or localization is found to be altered in specific disease states, providing targeted delivery opportunities.