KIF25 Antibody, FITC conjugated

Basic Research Questions

• What is KIF25 and what cellular functions does it perform?

  KIF25 (Kinesin Family Member 25) is a minus-end directed kinesin motor protein that plays a critical role in regulating centrosome dynamics. Research has shown that it functions as a tetrameric kinesin that suppresses centrosome separation during cell division. Without KIF25, premature centrosome separation occurs, with average distance between duplicated centrosomes increasing significantly in both interphase (from 2.44 ± 0.21 μm to 3.54 ± 0.25 μm) and prophase (from 8.57 ± 0.76 μm to 11.39 ± 0.69 μm) . KIF25 is also associated with microtubule organization and has been implicated in several cellular signaling pathways, particularly those involving cytoskeletal dynamics.

• What are the key specifications of commercially available KIF25 antibodies with FITC conjugation?

  Commercial FITC-conjugated KIF25 antibodies typically feature the following specifications:

  Characteristic	Specification
  Host Species	Rabbit (most common)
  Clonality	Polyclonal
  Reactivity	Human (primary target)
  Applications	ELISA, Immunofluorescence
  Form	Liquid
  Buffer	PBS with glycerol (typically 50%) and preservatives (e.g., 0.03% Proclin 300)
  Storage	-20°C to -80°C, avoid repeated freeze-thaw cycles
  Isotype	IgG

  For optimal results, most manufacturers recommend determining the ideal dilution empirically for each specific application .

• How should FITC-conjugated KIF25 antibodies be stored to maintain functionality?

  To preserve the fluorescence activity and binding capacity of FITC-conjugated KIF25 antibodies, storage at -20°C to -80°C is recommended. The antibodies are typically supplied in a buffer containing 50% glycerol to prevent freeze damage. It is critical to aliquot the antibody upon receipt to avoid repeated freeze-thaw cycles, which can dramatically reduce antibody performance . Additionally, storage should be in dark conditions as FITC is light-sensitive and can photobleach when exposed to light for extended periods. When working with the antibody, minimize light exposure by wrapping tubes in aluminum foil and working under reduced ambient lighting.

• What is the excitation/emission profile of FITC and how does this impact microscopy setup?

  FITC (Fluorescein Isothiocyanate) has an excitation maximum at approximately 495nm and an emission maximum at 519nm, producing a bright green fluorescence . For optimal imaging of FITC-conjugated KIF25 antibodies, microscopy setups should include:

  • A light source capable of providing 490-495nm excitation (typically an argon laser or LED)

  • A FITC/GFP filter set (excitation filter: 475-495nm, dichroic mirror: 505nm, emission filter: 510-550nm)

  • Minimal exposure times to reduce photobleaching

  When designing multi-color experiments, consider that FITC's emission spectrum may overlap with other green fluorophores, requiring careful compensation in flow cytometry or selection of spectrally distinct fluorophores for co-staining experiments.

• What sample preparation methods are recommended for KIF25-FITC antibody staining?

  For optimal staining with FITC-conjugated KIF25 antibodies, the following sample preparation protocol is recommended:

  1. Fix cells with 1% paraformaldehyde in cold (-20°C) methanol for 10 minutes

  2. For super-resolution microscopy, fix with 3.2% paraformaldehyde with 0.1% glutaraldehyde in PBS at 37°C for 10 minutes, followed by reduction with 0.1% sodium borohydride

  3. Block with 20% donkey serum or full-strength FBS for 1 hour to minimize background

  4. Apply the KIF25-FITC antibody at empirically determined dilutions (typically 1:50 to 1:200)

  5. Incubate overnight at 4°C in a humidified chamber protected from light

  6. Wash thoroughly with PBS containing 0.1% Tween-20

  7. Mount using an anti-fade mounting medium containing DAPI for nuclear counterstaining

Intermediate Research Questions

• How can I validate the specificity of my KIF25-FITC antibody for experimental applications?

  Validating antibody specificity is crucial for reliable research data. For KIF25-FITC antibodies, implement the following validation strategies:

  1. Genetic validation: Use siRNA knockdown of KIF25 (validated sequences: 5′-AGUGGAAGUUUACAAUAAU-3′ and 5′-CAGAGUGACUUAGGAAUUA-3′) to demonstrate reduced signal compared to control siRNA

  2. Peptide competition assay: Pre-incubate the antibody with synthetic KIF25 peptide (corresponding to the immunogen sequence) before staining to block specific binding

  3. Multiple antibody validation: Compare staining patterns with another KIF25 antibody recognizing a different epitope (e.g., N-terminal vs. C-terminal)

  4. Orthogonal validation: Correlate protein detection with mRNA expression using RT-PCR

  5. Recombinant protein controls: Use cells transfected with EGFP-KIF25 to confirm co-localization with antibody staining

  Document all validation results systematically, including microscopy settings, exposure times, and quantitative measurements of signal reduction in controls.

• What buffer systems and additives are compatible with FITC conjugation and subsequent immunofluorescence?

  When working with FITC-conjugated antibodies, buffer compatibility is critical for maintaining both antibody functionality and fluorophore integrity:

  Compatible additives include:

  • Tris buffer (up to 20mM)

  • Sodium chloride (0.15M)

  • BSA (up to 0.5%)

  • HEPES (50mM)

  • Gelatin (up to 0.1%)

  Additives to avoid include:

  • Nucleophiles like amino acids (e.g., glycine)

  • Blocking agents like ethanolamine

  • Thiols (DTT, mercaptoethanol)

  For immunofluorescence applications with KIF25-FITC antibodies, use 0.01M PBS (pH 7.4) containing 0.03% Proclin-300 as a preservative . For long-term storage, 50% glycerol should be included in the buffer system to prevent freeze damage .

• How can I optimize dual immunofluorescence studies combining KIF25-FITC with other cellular markers?

  For effective multi-color imaging of KIF25 along with other cellular components:

  1. Select compatible fluorophores: Pair FITC (green) with spectrally distinct fluorophores such as Cy5 for far-red emission . Avoid red fluorophores with significant spectral overlap.

  2. Sequential staining protocol:

     • Apply primary antibodies sequentially if both are from the same host species

     • For KIF25 co-localization with tubulin, use YL1/2 antibody against detyrosinated tubulin followed by Cy5-donkey anti-rat secondary

  3. Cross-reactivity controls:

     • Include single-stained controls to assess bleed-through

     • Use isotype controls for each primary antibody

  4. Microscope setup:

     • Adjust exposure times independently for each channel

     • Capture images sequentially rather than simultaneously

     • Apply spectral unmixing if available

  5. Quantitative co-localization analysis:

     • Use Pearson's correlation coefficient or Manders' overlap coefficient

     • Analyze using ImageJ with JACoP plugin or similar software

  This approach is particularly valuable for studying KIF25's interaction with centrosome components like pericentrin and tubulin .

• What approaches can be used to study KIF25's role in centrosome dynamics using FITC-conjugated antibodies?

  To investigate KIF25's function in centrosome regulation:

  1. Centrosome distance measurement:

     • Label centrosomes with pericentrin antibody and counterstain with KIF25-FITC

     • Measure inter-centrosomal distance in fixed cells at different cell cycle stages

     • Compare control vs. KIF25 knockdown cells (expected increase from 2.44 ± 0.21 μm to 3.54 ± 0.25 μm in interphase)

  2. Live-cell imaging approach:

     • Transfect cells with GFP-KIF25 and RFP-Pericentrin constructs

     • Perform time-lapse imaging through cell cycle phases

     • Quantify centrosome movement and separation dynamics

  3. Microtubule dependency assessment:

     • Treat cells with nocodazole to disrupt microtubules

     • Evaluate KIF25 localization before and after treatment

     • Assess ability of KIF25 overexpression to rescue centrosome separation phenotypes

  4. Functional rescue experiments:

     • Deplete endogenous KIF25 using siRNA

     • Reintroduce EGFP-KIF25 wild-type or mutant constructs

     • Measure restoration of normal centrosome separation distances

  These methodologies provide comprehensive insights into KIF25's mechanistic role in centrosome positioning and separation during cell division.

• How do I troubleshoot weak or high background signals when using KIF25-FITC antibodies?

  When encountering signal issues with KIF25-FITC antibody staining:

  For weak signals:

  1. Increase antibody concentration incrementally (test range from 1:200 to 1:50)

  2. Extend incubation time to overnight at 4°C

  3. Optimize fixation—test paraformaldehyde vs. methanol fixation

  4. Enhance epitope accessibility with antigen retrieval

  5. Use signal amplification systems (e.g., tyramide signal amplification)

  For high background:

  1. Increase blocking stringency (use 20% serum as recommended )

  2. Add 0.1-0.3% Triton X-100 to reduce non-specific binding

  3. Include additional washing steps (minimum 3x15 minutes)

  4. Pre-absorb antibody with cell/tissue lysate from species of interest

  5. Reduce antibody concentration and ensure proper storage conditions

  For autofluorescence issues:

  1. Include quenching step with 0.1% sodium borohydride after fixation

  2. Use Sudan Black B (0.1% in 70% ethanol) treatment

  3. Employ spectral imaging and linear unmixing if available

  Document all optimization steps systematically to determine the ideal protocol for your specific experimental system.

Advanced Research Questions

• What super-resolution microscopy techniques are most suitable for KIF25-FITC imaging, and what modifications to standard protocols are required?

  Super-resolution imaging of KIF25 requires specific adaptations:

  Technique	Advantages	Protocol Modifications
  STED (Stimulated Emission Depletion)	Good for co-localization studies	Use FITC-conjugated antibodies at higher concentration (1:50); reduce pixel size to 20-30nm
  SIM (Structured Illumination Microscopy)	Less phototoxic, good for thicker samples	Standard FITC protocols work; optimize exposure to minimize bleaching
  STORM/PALM	Highest resolution (10-20nm)	Require special buffer systems with oxygen scavengers and reducing agents

  For optimal super-resolution imaging:

  1. Use specialized fixation with 3.2% paraformaldehyde and 0.1% glutaraldehyde in PBS at 37°C

  2. Perform sodium borohydride (0.1%) reduction step post-fixation

  3. Use silane-coated coverslips for better sample adherence

  4. Post-fix with 0.2% glutaraldehyde after antibody labeling

  This approach has been successfully used to visualize the precise localization of KIF25 relative to centrosomal components, revealing detailed structural arrangements not visible with conventional microscopy.

• How can I quantitatively assess KIF25's association with microtubules and centrosomes using image analysis?

  For rigorous quantitative analysis of KIF25 localization:

  1. Colocalization measurement:

     • Perform triple staining with KIF25-FITC, pericentrin, and detyrosinated tubulin

     • Calculate Manders' overlap coefficient between KIF25 and centrosomal/microtubule markers

     • Use Costes method for statistical significance of colocalization

  2. Intensity profile analysis:

     • Draw line profiles through centrosomes and along microtubules

     • Quantify fluorescence intensity ratios of KIF25 to centrosomal markers

     • Compare profiles between control and experimental conditions

  3. Centrosome enrichment quantification:

     • Measure fluorescence intensity within a defined ROI centered on centrosomes

     • Calculate enrichment as ratio of centrosomal to cytoplasmic signal

     • Compare enrichment factors across cell cycle stages and treatments

  4. Automated detection and measurement:

     • Use CellProfiler or similar software for high-throughput analysis

     • Implement machine learning classification of KIF25 distribution patterns

     • Extract multiple parameters (intensity, area, shape factors) for multivariate analysis

  This quantitative approach enables detection of subtle changes in KIF25 localization and interaction with cellular structures under various experimental conditions.

• What experimental approaches can determine whether KIF25 forms complexes with other proteins at centrosomes?

  To investigate KIF25 protein interactions at centrosomes:

  1. Proximity Ligation Assay (PLA):

     • Combine KIF25 antibody with antibodies against potential interaction partners

     • Quantify PLA signals at centrosomes vs. cytoplasm

     • Compare signal patterns before and after treatments (e.g., EGF stimulation or dasatinib)

  2. FRET/FLIM analysis:

     • Tag KIF25 and candidate partners with appropriate fluorophore pairs

     • Measure FRET efficiency specifically at centrosomes

     • Calculate interaction distances based on FRET measurements

  3. Co-immunoprecipitation with centrosome fractionation:

     • Isolate centrosome-enriched fractions using sucrose gradient centrifugation

     • Perform co-IP with KIF25 antibodies followed by mass spectrometry

     • Validate interactions with reverse co-IP and Western blotting

  4. Super-resolution co-localization:

     • Use multi-color STORM imaging of KIF25-FITC with potential partners

     • Analyze spatial distribution at nanometer resolution

     • Create 3D reconstruction of protein complex architecture

  These complementary approaches provide both biochemical and spatial evidence for KIF25's interactions with other proteins at centrosomes, revealing potential regulatory mechanisms and functions.

• How can I design experiments to investigate the minus-end directed motor activity of KIF25 using FITC-labeled antibodies?

  To characterize KIF25's minus-end directed motor activity:

  1. In vitro microtubule gliding assays:

     • Immobilize purified KIF25 on glass surfaces

     • Add fluorescently labeled microtubules and ATP

     • Track microtubule movement direction and velocity

     • Confirm minus-end directionality using polarity-marked microtubules

  2. Single-molecule motility assays:

     • Label KIF25 with quantum dots or fluorescent nanoparticles

     • Observe movement along immobilized microtubules using TIRF microscopy

     • Measure step size, velocity, and run length

  3. Cell-based microtubule dynamics:

     • Express wild-type vs. motor-dead KIF25 mutants

     • Use live-cell imaging with EB1-GFP to track microtubule plus-ends

     • Quantify microtubule growth rates and centrosome-nucleation frequency

     • Correlate with KIF25-FITC antibody staining in fixed cells

  4. Force measurement using optical traps:

     • Attach KIF25-coated beads to optical tweezers

     • Measure forces generated during microtubule binding and movement

     • Compare force generation of wild-type vs. mutant KIF25

  These approaches provide comprehensive characterization of KIF25's motor properties and how they contribute to its role in suppressing centrosome separation .

• What methods can be used to study the dynamics of KIF25 during cell division using FITC-conjugated antibodies?

  To investigate KIF25 dynamics throughout cell division:

  1. Fixed-cell time course analysis:

     • Synchronize cells and fix at defined time points across mitosis

     • Stain with KIF25-FITC antibodies and cell cycle markers

     • Quantify KIF25 localization changes at centrosomes and spindle poles

     • Compare interphase (average centrosome distance 2.44 ± 0.21 μm) vs. prophase (8.57 ± 0.76 μm) localization patterns

  2. Correlative light and electron microscopy (CLEM):

     • Locate KIF25-FITC signals using fluorescence microscopy

     • Process same samples for electron microscopy

     • Map ultrastructural details to fluorescence patterns

  3. Cell cycle-specific perturbation:

     • Apply KIF25 siRNA knockdown combined with cell synchronization

     • Measure effects on centrosome separation at specific cell cycle transitions

     • Perform rescue experiments with cell cycle-regulated KIF25 expression

  4. Photobleaching techniques:

     • Express GFP-KIF25 and perform FRAP at centrosomes

     • Calculate recovery half-times across cell cycle stages

     • Compare dynamics during interphase vs. mitotic entry

     • Validate observations using immunofluorescence with KIF25-FITC antibodies

  These approaches provide insights into how KIF25's dynamic behavior relates to its function in regulating centrosome positioning during the cell division cycle.

Methodological Considerations

• What is the optimal protocol for conjugating FITC to purified anti-KIF25 antibodies in a research laboratory?

  For researchers wishing to perform their own FITC conjugation to anti-KIF25 antibodies:

  1. Required materials:

     • Purified anti-KIF25 antibody (1-4 mg/ml in compatible buffer)

     • FITC conjugation kit with FITC mix, modifier, and quencher reagents

     • Antibody concentration and purification kit if needed

  2. Step-by-step protocol:

     • Add 1 μl modifier reagent to each 10 μl of antibody solution

     • Mix gently by pipetting

     • Add the modified antibody directly to lyophilized FITC mix

     • Resuspend by gentle pipetting

     • Incubate in the dark at room temperature (20-25°C) for 3 hours

     • For optimal results, use 10-20 μg antibody for 10 μg FITC mix

  3. Buffer considerations:

     • Ensure antibody is in a buffer free of primary amines and thiols

     • Compatible buffers include PBS, HEPES, and Tris (up to 20mM)

     • Avoid buffers containing glycine, ethanolamine, DTT, or mercaptoethanol

  4. Storage of conjugate:

     • Add glycerol to 50% final concentration

     • Aliquot and store at -20°C

     • Protect from light and avoid repeated freeze-thaw cycles

  Following this protocol ensures optimal FITC labeling efficiency while maintaining antibody activity and specificity.

• How can I develop an immunofluorescence protocol to study KIF25's relationship with other kinesin family members?

  To investigate potential functional relationships between KIF25 and other kinesins:

  1. Sequential double immunofluorescence protocol:

     • First round: Apply KIF25-FITC antibody (1:100 dilution)

     • Wash extensively (3x15 minutes in PBS-T)

     • Second round: Apply antibody against other kinesin (e.g., KIF5B or KIF16B )

     • Use spectrally distinct secondary antibody (e.g., Cy5-conjugated)

  2. Controls for specificity:

     • Single antibody controls to assess bleed-through

     • Peptide competition controls for each kinesin antibody

     • siRNA knockdown validation for each target kinesin

  3. Co-expression analysis:

     • Express tagged versions of KIF25 and other kinesins

     • Perform FRET analysis to assess proximity/interaction

     • Validate with co-immunoprecipitation studies

  4. Functional interdependence studies:

     • Knockdown one kinesin and assess effects on localization of others

     • Look for compensatory mechanisms using quantitative image analysis

     • Analyze effects on shared processes (e.g., centrosome dynamics, microtubule organization)

  This approach can reveal whether KIF25 functions independently or cooperatively with other kinesin family members in regulating cellular processes.

• What experimental design would allow comprehensive characterization of KIF25's subcellular distribution across different cell types and conditions?

  For systematic analysis of KIF25 localization patterns:

  1. Cell panel characterization:

     • Prepare panel of cell lines representing different tissues and origins

     • Standardize fixation and immunostaining with KIF25-FITC antibodies

     • Create subcellular distribution atlas across cell types

  2. Quantitative image analysis pipeline:

     • Develop automated segmentation of cellular compartments

     • Measure KIF25-FITC signal intensity in each compartment

     • Calculate enrichment factors and distribution profiles

     • Apply machine learning for pattern recognition

  3. Perturbation matrix design:

     • Test KIF25 localization under systematically varied conditions:

       • Cell cycle synchronization (G1, S, G2, M)

       • Cytoskeletal disrupting agents (nocodazole, cytochalasin D)

       • Growth factor stimulation (e.g., EGF treatment)

       • Kinase inhibitors (e.g., dasatinib for Src inhibition)

  4. Correlation with functional outcomes:

     • Link KIF25 distribution patterns to phenotypic outcomes

     • Measure centrosome separation distances

     • Assess microtubule organization and dynamics

     • Evaluate cell division timing and accuracy

  This comprehensive approach generates a multidimensional dataset revealing how KIF25's subcellular distribution varies with cell type and condition, providing insights into its context-dependent functions.

• How can mass spectrometry be used in conjunction with KIF25-FITC antibodies to identify the protein interaction network of KIF25?

  To map the KIF25 interactome using complementary antibody-based and mass spectrometry approaches:

  1. Proximity-dependent labeling strategy:

     • Create KIF25-BioID or KIF25-APEX2 fusion constructs

     • Perform proximity labeling in living cells

     • Purify biotinylated proteins and analyze by LC-MS/MS

     • Validate key interactions using KIF25-FITC antibodies for co-localization

  2. Quantitative immunoprecipitation followed by MS:

     • Perform IP with anti-KIF25 antibodies

     • Use tandem mass tagged isobaric labeling (TMT10plex kit) for multiplexed analysis

     • Compare interactome across conditions (e.g., ±nocodazole, ±EGF stimulation)

     • Apply SAINT algorithm for high-confidence interaction filtering

  3. Cross-linking mass spectrometry (XL-MS):

     • Apply protein cross-linkers to stabilize KIF25 complexes

     • Digest and analyze by specialized XL-MS workflows

     • Map interaction interfaces at amino acid resolution

     • Model structural details of KIF25 complexes

  4. Validation pipeline:

     • Confirm key interactions using reciprocal co-IP

     • Perform PLA to verify proximity in intact cells

     • Use FITC-conjugated KIF25 antibodies for co-localization studies

     • Apply FRET to measure interaction distances

  This integrated approach provides a comprehensive view of KIF25's protein interaction network, identifying both stable and transient binding partners and their regulation under different cellular conditions.

• What approaches can determine the impact of post-translational modifications on KIF25 function using FITC-conjugated antibodies?

  To investigate how post-translational modifications regulate KIF25:

  1. PTM-specific antibody approach:

     • Develop or source antibodies specific to phosphorylated, acetylated, or ubiquitinated KIF25

     • Perform dual immunofluorescence with KIF25-FITC and PTM-specific antibodies

     • Quantify ratio of modified to total KIF25 under various conditions

  2. Mass spectrometry-based PTM mapping:

     • Immunoprecipitate KIF25 under different conditions

     • Perform targeted MS analysis for PTM identification and quantification

     • Map modifications to functional domains of KIF25

  3. Functional impact assessment:

     • Generate phosphomimetic and phospho-deficient KIF25 mutants

     • Evaluate impact on centrosome separation functions

     • Measure effects on microtubule binding and motor activity

     • Compare localization patterns using KIF25-FITC antibody staining

  4. Stimulus-response profiling:

     • Track changes in KIF25 modifications following:

       • Growth factor stimulation (e.g., EGF treatment)

       • Cell cycle progression

       • Kinase inhibitor treatments (e.g., dasatinib)

     • Correlate modification status with centrosome dynamics and KIF25 localization

  This systematic approach reveals how post-translational modifications dynamically regulate KIF25's molecular interactions, subcellular localization, and functional activities in different cellular contexts.