KIF2B Antibody

What is KIF2B and what cellular functions does it perform?

KIF2B (Kinesin Family Member 2B) is a kinesin-13 family protein that plays a crucial role in mitotic fidelity. It functions primarily to promote the correction of kinetochore-microtubule (k-MT) attachment errors during prometaphase of cell division. KIF2B is particularly important for maintaining chromosomal stability by preventing chromosome missegregation, which when persistent can lead to chromosomal instability (CIN) - a common feature of solid tumors . As a kinesin-13 protein, KIF2B has microtubule depolymerizing activity that is spatially and temporally regulated during the cell cycle to ensure proper chromosome segregation during mitosis .

How can I detect endogenous KIF2B in human cell lines?

Detection of endogenous KIF2B in human cell lines can be challenging due to its typically low expression levels. Researchers have found that standard immunoblotting of total cell extracts from cultured human cells (like HeLa or U2OS) often does not show detectable signal without overexpression . For successful detection, consider:

• Using immunoprecipitation to concentrate the protein before detection

• Employing antibodies specifically validated for the detection of endogenous levels of KIF2B protein, such as rabbit polyclonal antibodies

• Utilizing immunofluorescence with proper fixation methods - cold methanol fixation has been reported as effective for KIF2B antibodies

• Creating positive controls through transient expression of GFP-tagged KIF2B

The difficulty in detecting endogenous KIF2B without overexpression suggests its tight regulation and potentially low abundance in normal cells, which aligns with its specific functions during mitosis.

Which applications are most suitable for KIF2B antibody use in research?

Based on validated research applications, KIF2B antibodies can be effectively used in:

• Western Blotting (WB): Typically at dilutions of 1:500 - 1:2000 for detecting KIF2B in cell extracts

• Immunohistochemistry (IHC): At dilutions of 1:50 - 1:200 for tissue section analysis

• Immunofluorescence (IF): At dilutions of 1:50 - 1:200 for subcellular localization studies, particularly for mitotic structures

• Immunochromatography (IC): For protein detection and isolation

• Co-immunoprecipitation experiments: For studying protein-protein interactions involving KIF2B

• Mass spectrometry analysis: Following immunoprecipitation to study post-translational modifications

For optimal results, cold methanol fixation is recommended specifically for KIF2B immunofluorescence studies, while other fixatives like glutaraldehyde or paraformaldehyde may be more suitable for other target proteins when performing co-localization experiments .

What are the recommended protocols for immunoprecipitating KIF2B for downstream analysis?

For effective immunoprecipitation of KIF2B, researchers should follow these methodological steps:

• Cell preparation: Use approximately 10^7 cells expressing KIF2B (native or tagged)

• Cell lysis: Lyse cells in extraction buffer containing 2% SDS, 50 mM Tris-HCl (pH 6.8), 1 mM EDTA, 2 mM EGTA, 1 mM DTT, and phosphatase inhibitors (10 mM sodium fluoride, 10 mM sodium pyrophosphate)

• Heat treatment: Heat lysate to 100°C to ensure complete protein denaturation

• Clarification: Centrifuge at 13,000 × g for 15 minutes and collect the supernatant

• Dilution: Dilute the supernatant 8-fold with SDS-scavenging buffer (20 mM Tris-HCl, pH 7.4, 140 mM NaCl, 10 mM sodium pyrophosphate, 3.4% Triton X-100)

• Antibody incubation: Incubate with KIF2B-specific antibody (approximately 60 μg) at 4°C for 12 hours with gentle agitation

• Bead addition: Add 50 μl of protein A-conjugated agarose bead slurry and incubate for 2 hours at 4°C

• Washing: Perform 5 washes with buffer containing 50 mM HEPES (pH 7), 75 mM KCl, 1 mM MnCl2, 2 mM EGTA, 4 mM MgCl2, and 3 mM DTT

• Protein elution: Elute protein by boiling in SDS-PAGE sample buffer

• Reduction and alkylation: Reduce with 5 mM DTT at 55°C for 30 minutes and alkylate with 15 mM iodoacetamide at room temperature in the dark for 15 minutes prior to SDS-PAGE analysis

This protocol has been successfully used to isolate KIF2B for downstream applications including mass spectrometry analysis of phosphorylation sites.

How should KIF2B antibodies be validated before use in critical experiments?

Proper validation of KIF2B antibodies is essential before their use in critical experiments. A comprehensive validation approach should include:

• Positive control verification:

  • Use cells overexpressing GFP-tagged KIF2B to confirm antibody specificity

  • Perform co-localization studies between the antibody signal and GFP-KIF2B

• Immunoblot analysis:

  • Compare reactivity in control cells versus cells expressing tagged KIF2B

  • Observe predicted molecular weight (~105 kDa for GFP-KIF2B fusion protein)

• Application-specific validation:

  • For immunofluorescence: Test different fixation methods (cold methanol has been shown to work well for KIF2B antibodies)

  • For western blotting: Determine optimal antibody concentration (typically 1:500 - 1:2000)

• Negative controls:

  • Use pre-immune serum or non-specific IgG in parallel experiments

  • Include KIF2B-depleted samples (via siRNA) when possible

• Cross-reactivity assessment:

  • Test for cross-reactivity with other kinesin-13 family members (Kif2a and Kif2c/MCAK)

  • Verify species reactivity as claimed by the manufacturer (human, mouse, rat, etc.)

These validation steps ensure reliable results, especially important given the challenges in detecting endogenous KIF2B and its critical role in mitotic research.

What are the optimal fixation and staining protocols for visualizing KIF2B at kinetochores?

For optimal visualization of KIF2B at kinetochores during mitosis, researchers should follow these specific fixation and staining protocols:

• Cell extraction options:

  • For kinetochore visualization: Extract cells in calcium-containing buffer (100 mM PIPES, pH 6.8, 1 mM MgCl2, 0.1% Triton X-100, 1 mM CaCl2)

  • For microtubule preservation: Use microtubule-stabilizing buffer (4 M glycerol, 100 mM PIPES, pH 6.9, 1 mM EGTA, 5 mM MgCl2, and 0.5% Triton X-100)

• Fixation method:

  • Cold methanol fixation is specifically recommended for KIF2B antibody staining

  • Alternative fixatives for co-staining purposes:

    • 1% glutaraldehyde for microtubule and Hec1 staining

    • 3.5% paraformaldehyde for Kif2a and MCAK antibodies

• Antibody incubation:

  • Perform antibody incubations in TBS-BSA (10 mM Tris, pH 7.5, 150 mM NaCl, and 1% bovine serum albumin)

  • Use KIF2B antibody at dilutions of 1:50 - 1:200 for optimal signal-to-noise ratio

• Co-staining markers:

  • Include kinetochore markers such as CREST antibody to confirm kinetochore localization

  • Consider microtubule staining with tubulin-specific antibodies (e.g., DM1α)

  • For prometaphase-specific localization, co-stain with prometaphase markers

• Controls for specificity:

  • Include cells expressing GFP-KIF2B as positive controls

  • Use cells depleted of KIF2B as negative controls

This protocol has been validated for studying the temporal and spatial regulation of KIF2B at kinetochores during different stages of mitosis.

How does phosphorylation regulate KIF2B activity during mitosis?

Phosphorylation plays a critical role in regulating both the localization and activity of KIF2B during mitosis. Mass spectrometry analysis has identified multiple phosphorylation sites on KIF2B, with some being specifically regulated by Polo-like kinase 1 (Plk1) .

Key phosphorylation sites and their functions:

• Threonine 125 (T125):

  • Directly phosphorylated by Plk1

  • Required for KIF2B activity in the correction of kinetochore-microtubule (k-MT) attachment errors

  • Does not affect localization but modulates enzymatic activity

• Serine 204 (S204):

  • Directly phosphorylated by Plk1

  • Required for the kinetochore localization of KIF2B specifically during prometaphase

  • Critical for proper spatial positioning of KIF2B at its site of action

• Other phosphorylation sites:

  • Multiple additional sites have been identified by mass spectrometry

  • Some sites show differential phosphorylation between prometaphase and metaphase

  • Certain sites are acutely sensitive to inhibition of Plk1 or Aurora kinases

This phosphoregulation mechanism explains how KIF2B activity is temporally restricted to prometaphase, where it plays a crucial role in correcting erroneous k-MT attachments to prevent chromosome missegregation and maintain genomic stability.

What is the relationship between KIF2B and chromosomal instability (CIN) in cancer cells?

The relationship between KIF2B and chromosomal instability (CIN) in cancer cells is multifaceted and involves KIF2B's role in maintaining proper chromosome segregation:

• Prevention of k-MT attachment errors:

  • KIF2B promotes the correction of kinetochore-microtubule (k-MT) attachment errors specifically during prometaphase

  • These errors, if uncorrected, lead to lagging chromosomes in anaphase - the most common cause of CIN

• Temporal regulation of error correction:

  • KIF2B activity is restricted to prometaphase through Plk1-dependent phosphorylation

  • This temporal regulation ensures proper chromosome alignment before anaphase onset

• Connection to solid tumors:

  • Solid tumors are frequently aneuploid (have abnormal chromosome numbers)

  • Many display high rates of ongoing chromosome missegregation (CIN)

  • Defects in k-MT attachment error correction pathways, potentially including KIF2B dysfunction, can contribute to CIN

• Mitotic fidelity:

  • KIF2B functions to ensure mitotic fidelity through its microtubule depolymerizing activity

  • Proper regulation of this activity is essential for preventing chromosome missegregation

Understanding the precise role of KIF2B in preventing CIN provides insights into potential therapeutic approaches for cancers characterized by chromosomal instability, as well as basic mechanisms of maintaining genomic stability in normal cells.

How does KIF2B functionally differ from other kinesin-13 family members (Kif2a and MCAK/Kif2c)?

The kinesin-13 family includes three members in humans: Kif2a, Kif2b, and Kif2c/MCAK. While they share similar catalytic domains and microtubule-depolymerizing activities, they exhibit distinct functions and regulations:

These distinct characteristics reflect their specialized functions in ensuring proper chromosome segregation during cell division. While all three proteins contribute to genomic stability, KIF2B's function appears more specifically restricted to the prometaphase correction of k-MT attachment errors, which is essential for preventing chromosome missegregation .

Why is endogenous KIF2B difficult to detect in many cell lines, and how can this be overcome?

Endogenous KIF2B is notoriously difficult to detect in many cell lines due to several factors:

• Low abundance:

  • KIF2B is typically expressed at low levels in cultured human cells

  • Standard immunoblots of total cell extracts often show no detectable signal without overexpression

• Cell cycle-specific expression:

  • KIF2B function is primarily restricted to prometaphase of mitosis

  • Expression or stability may be tightly regulated throughout the cell cycle

• Technical challenges in antibody generation:

  • The unique N-terminal domain used for antibody generation (~143 amino acids) may have limited immunogenicity

  • Antibody affinity might be insufficient for detecting low levels of the protein

Strategies to overcome detection challenges:

• Enrichment approaches:

  • Synchronize cells in mitosis using nocodazole or other mitotic arrestors to increase the proportion of cells expressing KIF2B

  • Use immunoprecipitation to concentrate the protein before detection

  • Fractionate samples to isolate mitotic structures like kinetochores

• Signal amplification:

  • Employ more sensitive detection methods such as chemiluminescence with extended exposure times

  • Use tyramide signal amplification for immunofluorescence detection

  • Consider super-resolution microscopy techniques for localization studies

• Alternative validation approaches:

  • Create positive controls through transient expression of tagged KIF2B

  • Use mRNA detection methods (qPCR, RNA-seq) to confirm expression

  • Apply CRISPR tagging of endogenous KIF2B with bright fluorescent proteins

Understanding these challenges is crucial for researchers to properly design experiments and interpret results when studying endogenous KIF2B function.

What are the most common pitfalls in mass spectrometry analysis of KIF2B phosphorylation sites?

Mass spectrometry analysis of KIF2B phosphorylation sites presents several technical challenges that researchers should be aware of:

• Sample preparation issues:

  • Insufficient enrichment of KIF2B due to low endogenous expression levels

  • Incomplete digestion by trypsin can lead to missed phosphorylation sites

  • Phosphopeptide loss during sample preparation due to their hydrophilic nature

• Phosphopeptide enrichment challenges:

  • Suboptimal enrichment efficiency for KIF2B phosphopeptides

  • Bias toward certain phosphopeptides based on charge or hydrophobicity

  • Competition from highly abundant phosphoproteins in the sample

• Mass spectrometry detection limitations:

  • Incomplete fragmentation of phosphopeptides leading to ambiguous site localization

  • Suppression of phosphopeptide signals by non-phosphorylated peptides

  • Limited sensitivity for detecting low-abundance phosphorylation events

• Data analysis complexities:

  • False localization of phosphorylation sites within peptides containing multiple S/T/Y residues

  • Difficulties in quantifying changes in phosphorylation stoichiometry

  • Challenges in distinguishing biological variability from technical variability

Best practices to address these challenges:

• Use SILAC or other quantitative approaches to compare phosphorylation states between conditions

• Employ specific phosphopeptide enrichment strategies (TiO2, IMAC, phospho-antibodies)

• Apply appropriate statistical methods and site localization algorithms

• Validate key phosphorylation sites through site-directed mutagenesis and functional assays

• Consider using multiple proteases beyond trypsin to increase phosphosite coverage

The research by Manning et al. successfully employed SILAC labeling and mass spectrometry to identify Plk1-dependent phosphorylation sites on KIF2B, demonstrating that these challenges can be overcome with proper methodology .

How can researchers distinguish between the effects of KIF2B depletion and potential off-target effects in functional studies?

Distinguishing between specific KIF2B depletion effects and potential off-target effects in functional studies requires rigorous experimental design and multiple validation approaches:

• Rescue experiments:

  • Express RNAi/CRISPR-resistant KIF2B variants to restore function in depleted cells

  • Use both wild-type KIF2B and specific phospho-mutants (e.g., T125A, S204A) to validate site-specific functions

  • Ensure expression levels are comparable to endogenous protein to avoid overexpression artifacts

• Multiple depletion strategies:

  • Use different siRNA/shRNA sequences targeting distinct regions of KIF2B mRNA

  • Compare siRNA results with CRISPR/Cas9 knockout approaches

  • Apply different inducible depletion systems (e.g., degron tags, auxin-inducible degradation)

• Specificity controls:

  • Monitor effects on other kinesin-13 family members (Kif2a and MCAK/Kif2c) to ensure they're not affected

  • Use closely related kinesins as controls for specificity of phenotypes

  • Perform transcriptome or proteome analysis to identify potential off-target effects

• Phenotypic analysis pipeline:

  • Establish clear quantitative metrics for phenotypic assessment (e.g., lagging chromosome frequency, mitotic timing)

  • Utilize live-cell imaging to track dynamics rather than relying solely on fixed-cell analysis

  • Apply computational image analysis to reduce observer bias

• Functional complementation tests:

  • Determine whether other kinesin-13 proteins can compensate for KIF2B loss

  • Test if KIF2B can rescue phenotypes of other kinesin-13 depletions

  • Analyze synthetic phenotypes from combined depletions

By implementing these rigorous validation approaches, researchers can confidently attribute observed phenotypes specifically to KIF2B function rather than to off-target effects or general perturbation of microtubule dynamics.

What are the emerging techniques for studying KIF2B dynamics and regulation in live cells?

Several cutting-edge techniques are emerging for studying KIF2B dynamics and regulation in live cells, offering new insights beyond traditional fixed-cell approaches:

• Advanced live-cell imaging approaches:

  • FRAP (Fluorescence Recovery After Photobleaching) to measure KIF2B turnover at kinetochores

  • Single-molecule tracking to follow individual KIF2B molecules during mitosis

  • Lattice light-sheet microscopy for high-resolution 3D imaging with reduced phototoxicity

  • Super-resolution techniques (PALM/STORM) adapted for live-cell visualization

• Biosensor technologies:

  • FRET-based sensors to detect KIF2B conformational changes upon phosphorylation

  • Phospho-specific sensors to visualize Plk1-mediated phosphorylation events in real-time

  • Optogenetic tools to manipulate KIF2B activity with spatiotemporal precision

• Genome editing applications:

  • CRISPR/Cas9 knock-in of fluorescent tags at the endogenous KIF2B locus

  • Creation of specific phosphomutants at endogenous loci using base editing

  • Auxin-inducible degron tags for rapid protein depletion studies

  • Split-GFP complementation to visualize protein-protein interactions in vivo

• Advanced biochemical approaches:

  • Proximity labeling techniques (BioID, APEX) to identify KIF2B interactors in specific cellular compartments

  • Time-resolved mass spectrometry to track phosphorylation dynamics throughout mitosis

  • Cryo-electron microscopy to determine structural changes upon phosphorylation

These emerging techniques promise to provide unprecedented insights into the temporal and spatial regulation of KIF2B activity during mitosis, potentially revealing new regulatory mechanisms and interactors that could be targeted for therapeutic intervention in diseases characterized by chromosomal instability.

How might KIF2B function be targeted for potential therapeutic applications in cancer?

Given KIF2B's role in maintaining chromosomal stability and preventing aneuploidy, it represents a potential therapeutic target for cancer treatment through several potential approaches:

• Direct targeting strategies:

  • Small molecule inhibitors of KIF2B ATPase or microtubule-binding activity

  • Allosteric modulators that prevent Plk1-mediated activation

  • Degraders (PROTACs) to selectively remove KIF2B protein from cancer cells

  • Peptide inhibitors that disrupt specific protein-protein interactions

• Synthetic lethality approaches:

  • Identify contexts where KIF2B inhibition is selectively lethal to cancer cells

  • Target KIF2B in combination with taxanes or other microtubule-targeting agents

  • Exploit dependencies on KIF2B in cells with CIN or specific oncogenic drivers

  • Combine with inhibitors of the spindle assembly checkpoint

• Biomarker development:

  • Use KIF2B expression or phosphorylation status as predictive biomarkers for response to anti-mitotic therapies

  • Develop diagnostics to identify patients with tumors dependent on KIF2B function

  • Monitor chromosomal instability as a pharmacodynamic marker of KIF2B inhibition

• Rational combination strategies:

  • Pair KIF2B inhibition with Plk1 inhibitors to enhance mitotic catastrophe in cancer cells

  • Combine with inhibitors of centrosome clustering to selectively target cells with supernumerary centrosomes

  • Target parallel pathways of k-MT error correction to overcome potential resistance mechanisms

• Cell cycle-specific delivery approaches:

  • Develop mitosis-specific drug delivery systems to minimize effects on non-dividing cells

  • Create prodrugs activated specifically during mitosis

  • Utilize tumor-targeting strategies to limit systemic toxicity

The therapeutic potential of targeting KIF2B lies in its specific role during mitosis and its connection to chromosomal instability, which is a hallmark of many aggressive cancers but rare in normal tissues, potentially providing a therapeutic window.