KIF25 Antibody

What is KIF25 and why are antibodies against it important for cell biology research?

KIF25 (Kinesin Family Member 25) is a tetrameric kinesin protein that functions as a bipolar, minus-end directed motor protein. Its primary biological role is to suppress centrosome separation during interphase by tethering centrosomes together through microtubule-dependent mechanisms . This function is critical for maintaining proper nuclear positioning and ensuring stable spindle orientation at the onset of mitosis.

KIF25 antibodies are valuable research tools because they enable:

• Visualization of centrosome dynamics during cell cycle progression

• Investigation of microtubule-dependent nuclear positioning

• Analysis of spindle orientation mechanisms

• Study of cell division aberrations in disease models

The importance of KIF25 can be understood through its knockdown effects: when KIF25 is depleted, cells exhibit premature centrosome separation during interphase (increasing from 2.44 ± 0.21 μm in control cells to 3.54 ± 0.25 μm in KIF25 knockdown cells) . This leads to microtubule-dependent nuclear translocation and disruption of cortical spindle positioning machinery.

What types of KIF25 antibodies are currently available for research applications?

Several types of KIF25 antibodies have been developed for research applications:

These antibodies offer researchers flexibility in experimental design based on specific requirements such as species cross-reactivity, epitope accessibility, and application compatibility .

How can I validate the specificity of a KIF25 antibody?

Validating antibody specificity is critical for ensuring reliable research outcomes. For KIF25 antibodies, consider these methodological approaches:

• Genetic knockout/knockdown controls:

  • Utilize KIF25 siRNA knockdown or CRISPR/Cas9 knockout cells as negative controls

  • Research has validated antibody specificity by confirming the absence of ~220 kDa bands in KIF25 knockout or knockdown MEF lysates compared to wild-type

• Recombinant protein expression validation:

  • Express KIF25 with epitope tags in cells lacking endogenous KIF25

  • Verify co-localization of antibody signal with the tagged protein

• Western blot analysis:

  • Confirm single band detection at the expected molecular weight (~220 kDa)

  • Compare multiple antibodies targeting different epitopes

• Immunoprecipitation followed by mass spectrometry:

  • Verify that KIF25 is the primary protein pulled down by the antibody

• Cross-reactivity assessment:

  • Test against closely related kinesin family proteins to ensure specificity

Example validation protocol from published research: "We found that the anti-Kif26b antibodies recognized protein bands at the predicted size of Kif26b (~220 kD) in wild-type MEF lysates but not in the Kif26b knockout or knockdown MEF lysates, confirming that our antibodies specifically recognize endogenous Kif26b" .

How can I optimize KIF25 antibody protocols for immunofluorescence microscopy of centrosome dynamics?

Optimizing KIF25 antibody protocols for high-quality immunofluorescence imaging of centrosome dynamics requires attention to several methodological details:

Fixation and Permeabilization:

• For standard resolution: Fix cells in 1% PFA in -20°C methanol for 10 minutes, followed by blocking with 20% donkey serum for 1 hour

• For super-resolution imaging: Fix cells in 37°C PBS containing 3.2% paraformaldehyde and 0.1% glutaraldehyde for 10 minutes, then reduce with 0.1% Sodium Borohydride before blocking with full-strength FBS

Antibody Dilutions and Incubation:

• Primary antibody: Use at 1:100-1:200 dilution for monoclonal antibodies like 2C9

• For IHC applications: Sigma's HPA046153 antibody performs optimally at 1:200-1:500 dilution

• Extend primary antibody incubation to overnight at 4°C for improved signal-to-noise ratio

Co-labeling Strategy:

• Pair KIF25 antibody with pericentrin/γ-tubulin antibody for precise centrosome localization

• Include α-tubulin labeling to visualize microtubule networks

Image Acquisition Parameters:

• Use confocal microscopy with z-stacks (0.2-0.3 μm steps) for accurate centrosome distance measurements

• For live-cell imaging applications, consider using recombinant single-chain variable fragments derived from KIF25 antibodies

Quantification Method:

• Measure inter-centrosomal distance using line scan analysis

• Normal interphase centrosome separation typically ranges from 2.44 ± 0.21 μm in control cells

What are the considerations for using KIF25 antibodies in tracking disease-associated protein dynamics?

KIF25 antibodies can be valuable tools for studying disease-associated protein dynamics, particularly in conditions affecting cellular organization and division. For optimal results:

• Disease-specific considerations:

  • In neurodegenerative disease models: Recombinant H2 kinesin antibodies have successfully detected ALS-associated KIF5A aggregates that conventional fluorescent protein tagging might miss

  • Apply similar principles to detect potential KIF25 aggregates or mislocalization in disease states

• Sensitivity enhancement strategies:

  • Signal amplification: Consider tyramide signal amplification (TSA) to detect low-abundance KIF25 in patient samples

  • Antibody combinations: Use multiple antibodies targeting different epitopes to enhance detection reliability

• Quantitative analysis approaches:

  • Develop automated image analysis workflows for unbiased quantification

  • Establish clear thresholds for abnormal KIF25 distribution based on control samples

• Experimental controls for disease studies:

  • Include age-matched controls when studying age-related conditions

  • Use isotype controls to distinguish specific binding from background

  • Incorporate genetic models with known KIF25 alterations as reference points

• Tissue-specific protocol adaptations:

  • Brain tissue: Extended fixation time and specialized antigen retrieval

  • Muscle tissue: Additional permeabilization steps may be required

  • Paraffin sections: Optimal IHC dilutions range from 1:150-1:500 depending on the antibody

How can I apply computational modeling approaches to analyze KIF25 antibody binding characteristics?

Recent advances in computational biology offer powerful approaches to analyze and predict antibody-antigen interactions. For KIF25 antibody research:

• Binding mode analysis techniques:

  • Utilize biophysics-informed models that associate distinct binding modes with specific ligands

  • These approaches have been successfully applied to disentangle multiple binding modes in antibody research

• Structural analysis of paratope-epitope interactions:

  • Analysis of over 850,000 atom-atom contacts from antibody-antigen complexes reveals patterns in amino acid frequencies that can be applied to KIF25 antibody design

  • Key findings show that sdAbs (single-domain antibodies) employ different mechanisms to compensate for their smaller size

• Machine learning applications:

  • Predictive models can inform antibody engineering to improve specificity

  • In silico antibody evolution techniques developed at Stanford use directed evolution for predicting improved antibody variants

• Validation approach:

  • Experimental validation through site-directed mutagenesis of predicted key residues

  • Affinity measurements before and after computational optimization

What controls should be implemented when using KIF25 antibodies in knockdown experiments?

Robust experimental controls are essential when using KIF25 antibodies in knockdown studies to ensure reliable interpretation of results:

• Genetic controls:

  • Negative control siRNA: Use non-targeting control siRNA (e.g., Ambion negative control siRNA1) processed identically to KIF25 siRNA samples

  • Multiple siRNA constructs: Validate phenotypes with at least two independent siRNAs targeting different regions of the KIF25 gene to rule out off-target effects

  • Published example: "For Kif25 depletion, two siRNAs targeted to distinct regions of the Kif25 gene were used independently to assess Kif25 KD phenotypes (targeting sequences (1) 5′-AGUGGAAGUUUACAAUAAU-3′ and (2) 5′-CAGAGUGACUUAGGAAUUA-3′)"

• Expression rescue controls:

  • Express siRNA-resistant KIF25 to rescue the knockdown phenotype

  • Example: "Rescue of Kif25 knockdown by EGFP-Kif25 expression restores control-like interphase centrosome separation distances (1.984 ± 0.21 μm, P=0.036 compared to control)"

• Antibody validation in knockdown context:

  • Confirm reduction of KIF25 signal via:

    • Western blot (quantitative band intensity reduction)

    • Immunofluorescence (reduced staining intensity)

  • Include signal quantification relative to loading control or housekeeping protein

• Timing considerations:

  • Monitor KIF25 depletion timeline (typically 36-48 hours post-transfection)

  • Perform phenotypic analyses at timepoints with confirmed protein reduction

• Combinatorial knockdowns:

  • For studies requiring depletion of multiple kinesin family members, consider miRNA-based knockdown systems similar to those used for KIF5A/B/C triple knockdown

  • Validate specificity of remaining kinesin detection using appropriate antibodies

How can recombinant KIF25 antibodies be engineered for specialized research applications?

Recombinant antibody technology offers opportunities to customize KIF25 antibodies for specialized applications:

• Single-chain variable fragment (scFv) development:

  • Convert conventional KIF25 antibodies to scFv format for applications requiring smaller size

  • Methodology: Determine VH and VL sequences, connect with flexible linker, express in appropriate system

  • Similar approaches with kinesin antibodies have shown that scFvs can specifically recognize targets in cells

• Species diversification:

  • Engineer "chickenized" or other species variants to expand compatibility with multiple detection systems

  • This approach broadens applications in immunofluorescence microscopy by enabling multi-color imaging

• Functional modifications:

  • Create blocking antibodies by targeting functional domains of KIF25

  • Develop conformation-specific antibodies to distinguish active vs. inactive states

• Fusion protein strategies:

  • Generate fluorescent protein fusions for live-cell imaging

  • Create intrabody versions for tracking KIF25 in living cells

  • Engineering challenge: "While we have not yet succeeded in doing this with the H2 mAb, the scFv can be used as a genetically engineered probe to label endogenous antigens in the cell. This intrabody technique can be used to inhibit the function of antigens in the cell"

• Expression system considerations:

  • HEK293/293FT cells provide cost-effective expression systems for recombinant antibodies

  • For some applications, purification may not be required: "The experiments described in this report were performed using the cultured medium because we found that purification was not required for western blotting or immunofluorescence microscopy"

What are common issues with KIF25 antibody experiments and how can they be resolved?

For KIF25-specific considerations:

• If detecting centrosome separation dynamics, remember that nocodazole treatment promotes interphase centrosome separation that cannot be rescued by KIF25 overexpression

• For enhanced specificity in Western blots, recommended antibody dilutions range from 1:2000 for monoclonal antibodies to 0.04-0.4 μg/mL for polyclonal antibodies

How can I optimize KIF25 antibody protocols for different experimental techniques?

For Western Blotting:

• Protein extraction: Use RIPA buffer with protease inhibitors

• Protein loading: 20-40 μg total protein per lane

• Antibody dilutions: 1:2000 for monoclonal (ABIN1499039) , 0.04-0.4 μg/mL for polyclonal (HPA046153)

• Signal detection: Enhanced chemiluminescence with exposure time optimization

For Immunohistochemistry:

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Antibody dilutions: 1:200-1:500 for HPA046153 , 1:150 for IHC-P applications of ABIN1499039

• Detection system: DAB chromogen with hematoxylin counterstain

• Controls: Include isotype control and known positive tissue sections

For Immunofluorescence:

• Fixation: 1% PFA in -20°C methanol for standard applications

• Specialized fixation for super-resolution: 3.2% paraformaldehyde with 0.1% glutaraldehyde

• Antibody dilution: 1:100 for IF with monoclonal antibodies like 2C9

• Nuclear counterstain: DAPI at 1:10,000 dilution

For Co-immunoprecipitation:

• Lysis buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate with protease inhibitors

• Antibody amount: 2-5 μg per 500 μg of total protein

• Pre-clearing: Incubate lysate with protein A/G beads before adding antibody

• Controls: Include IgG control and input sample

What are the latest methodological advances in KIF25 antibody applications for studying centrosome dynamics?

Recent advances in KIF25 antibody applications have expanded capabilities for studying centrosome dynamics:

• Super-resolution imaging techniques:

  • Sample preparation: Specialized fixation with 3.2% paraformaldehyde and 0.1% glutaraldehyde

  • Co-labeling strategy: GFP-Kif25 and RFP-Pericentrin for protein localization combined with YL1/2 antibody against detyrosinated tubulin

  • This approach enables nanoscale resolution of KIF25 localization relative to centrosome structures

• Live-cell imaging approaches:

  • Development of recombinant scFv antibody fragments that specifically recognize kinesins in cells

  • These can be adapted for KIF25 to study dynamic centrosome movements without fixation artifacts

• Quantitative analysis methods:

  • Automated tracking of centrosome separation using machine learning algorithms

  • Statistical approaches to correlate centrosome separation and spindle orientation:
    "In control cells the majority of the population display normal centrosome separation and form a properly oriented parallel spindle (grey bars, 66% of cells)"

• Genetic modification strategies:

  • miRNA-based knockdown systems that can simultaneously target multiple kinesin family members

  • CRISPR/Cas9 knockout combined with rescue experiments using modified KIF25 constructs

• Computational modeling integration:

  • Machine learning approaches to predict antibody binding characteristics

  • Structural analysis of antibody-antigen interfaces to understand specificity determinants

By combining these methodological advances, researchers can gain unprecedented insights into the role of KIF25 in regulating centrosome dynamics and nuclear positioning.

How might KIF25 antibodies contribute to understanding disease mechanisms?

The role of KIF25 in maintaining proper nuclear positioning and spindle orientation suggests potential implications in disease contexts where these processes are dysregulated:

• Cancer research applications:

  • Investigate KIF25 expression and localization in cancer cells with aberrant cell division

  • Develop KIF25 antibodies as potential biomarkers for tumors with centrosome amplification

  • Research question: Does KIF25 dysregulation contribute to chromosomal instability in cancer?

• Neurodevelopmental disorders:

  • Examine KIF25 function in neuronal migration and brain development

  • Apply lessons from kinesin-related disorders such as ALS-associated KIF5A mutations

  • Could KIF25 antibodies help identify abnormal centrosome dynamics in neurodevelopmental conditions?

• Methodological advances needed:

  • Development of phospho-specific KIF25 antibodies to monitor regulation

  • Creation of conformation-specific antibodies to distinguish active vs. inactive states

  • Integration with emerging spatial transcriptomics technologies

• Therapeutic implications:

  • Potential for developing function-blocking antibodies against KIF25 for therapeutic applications

  • Application of machine learning models for in silico antibody evolution to create highly specific KIF25-targeting molecules

How can emerging antibody technologies be applied to KIF25 research?

The field of antibody technology is rapidly evolving, offering exciting possibilities for KIF25 research:

• Nanobody and single-domain antibody applications:

  • Development of camelid-derived nanobodies against KIF25 for super-resolution microscopy

  • Advantages: smaller size (~15 kDa) allows better tissue penetration and epitope access

  • Research findings show that single-domain antibodies employ different mechanisms to compensate for their smaller size when compared to conventional antibodies

• Bispecific antibody development:

  • Create bispecific antibodies targeting KIF25 and other centrosome components

  • Applications: co-localization studies, protein-protein interaction analysis

• Antibody engineering through computational approaches:

  • Apply machine learning models to predict improved KIF25 antibody variants

  • Use structural data from antibody-antigen complexes to inform design strategies

  • "Predicting a small, manageable set (~10) of high-likelihood protein variants from a single antibody sequence using the predictive capabilities of protein language models"

• Intrabody approaches:

  • Develop genetically encoded antibody fragments for tracking endogenous KIF25 in living cells

  • Challenge: "The scFv is often not soluble in cytosol. Fusion with superfolder GFP (sfGFP) or the ultra-stable cytoplasmic antibody (STAND) could be used to increase the folding and solubility of scFv in the cytoplasm"

• Integration with emerging technologies:

  • CRISPR-based proximity labeling combined with KIF25 antibodies

  • Spatial transcriptomics to correlate KIF25 protein localization with gene expression patterns

By leveraging these emerging antibody technologies, researchers can gain unprecedented insights into KIF25 biology and its role in cellular processes.