KIF22 Antibody, FITC conjugated

What is KIF22 and why is it significant in cellular research?

KIF22 is a kinesin family member involved in spindle formation and chromosomal movement during cell division. It binds to both microtubules and DNA, playing a critical role in mitotic and meiotic processes. Recent research has demonstrated that KIF22 contributes to the congression of laterally attached chromosomes in NDC80-depleted cells and generates forces that contribute to mitotic chromosome congression and alignment . KIF22's significance in research extends to its involvement in various pathological conditions, as its overexpression has been linked to poor prognosis in multiple myeloma and gastric cancer patients .

What are the available types of KIF22 antibodies for research applications?

Researchers can select from several types of KIF22 antibodies based on their experimental needs. Polyclonal rabbit antibodies are commonly available with reactivity to human and mouse samples . Monoclonal rabbit antibodies are also available for applications requiring higher specificity, particularly for Western blotting with human samples . The choice between polyclonal and monoclonal antibodies should be guided by the specific research application, with polyclonal antibodies offering broader epitope recognition while monoclonals provide higher specificity to a single epitope.

What is FITC conjugation and how does it benefit KIF22 research?

FITC (Fluorescein isothiocyanate) is a green fluorescent dye with excitation and emission wavelengths of 495nm and 519nm, respectively. Conjugating FITC to KIF22 antibodies enables direct visualization of KIF22 localization within cells without requiring secondary antibodies. This approach is particularly valuable for fluorescence microscopy, flow cytometry, and multiplexed immunofluorescence studies where multiple proteins need to be visualized simultaneously . FITC conjugation reduces background signal and minimizes cross-reactivity issues that can occur with secondary antibody detection systems.

What experimental applications are suitable for FITC-conjugated KIF22 antibodies?

FITC-conjugated KIF22 antibodies are primarily suited for immunofluorescence applications, including immunocytochemistry (ICC), immunohistochemistry (IHC) of frozen sections, flow cytometry, and high-content screening. These antibodies are particularly valuable for co-localization studies with proteins detected using antibodies conjugated to fluorophores with different spectral properties. While unconjugated KIF22 antibodies are suitable for Western blotting, immunohistochemistry, and immunofluorescence , FITC-conjugated versions are optimized for direct detection in fluorescence-based applications.

How should researchers prepare samples for optimal KIF22-FITC antibody binding?

For optimal KIF22-FITC antibody binding, researchers should:

• Fix cells or tissues using 4% paraformaldehyde to preserve cellular structure while maintaining antigenicity

• Permeabilize with 0.1-0.5% Triton X-100 for intracellular access (KIF22 is primarily localized to the nucleus during interphase and associates with chromosomes and spindle apparatus during mitosis)

• Block with serum (10%) to reduce non-specific binding

• For tissue sections, perform antigen retrieval as KIF22 epitopes may be masked during fixation

• Optimize antibody concentration through titration experiments (typical starting dilutions are 1:100 to 1:500)

• Include appropriate negative controls (secondary antibody alone) and positive controls (cell lines known to express KIF22)

These preparations ensure specific labeling while minimizing background fluorescence.

What is the recommended protocol for conjugating KIF22 antibodies with FITC?

For researchers performing their own FITC conjugation of KIF22 antibodies, the following protocol is recommended:

• Ensure the antibody is highly purified and in an appropriate buffer free of primary amines and thiols which can interfere with the conjugation chemistry

• Adjust antibody concentration to 0.5-5 mg/ml for optimal results

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

• Add the antibody-modifier mixture directly to lyophilized FITC mix

• Incubate in the dark at room temperature (20-25°C) for 3 hours (incubation can be extended overnight without negative effects)

• Add quencher reagent if the kit includes one

This protocol typically yields antibodies with a fluorophore-to-protein ratio of 4-7 FITC molecules per antibody, providing bright signal while maintaining antibody specificity and affinity.

What controls should be included when using FITC-conjugated KIF22 antibodies?

When designing experiments with FITC-conjugated KIF22 antibodies, the following controls are essential:

How can FITC-conjugated KIF22 antibodies be used to study cell cycle-dependent localization?

KIF22 exhibits dynamic subcellular localization throughout the cell cycle, making it an excellent target for cell cycle studies. For investigating this dynamic localization:

• Synchronize cells at different cell cycle stages using established methods (double thymidine block for G1/S, nocodazole for G2/M)

• Fix and stain cells with FITC-conjugated KIF22 antibodies

• Co-stain with markers for specific cell cycle phases (e.g., phospho-histone H3 for mitosis)

• Use confocal microscopy to track KIF22 localization changes from nuclear in interphase to chromosome-associated during mitosis

This approach reveals KIF22's chromosome binding and spindle formation functions during the G2/M phase, correlating with its role in cell cycle regulation via CDC25C/CDK1/cyclinB1 pathway . Time-lapse imaging with stably transfected cell lines expressing FITC-conjugated anti-KIF22 can provide even more detailed insights into dynamic relocalization.

What methodologies are suitable for studying KIF22's role in pathological conditions using FITC-conjugated antibodies?

To investigate KIF22's role in cancer and other pathological conditions:

• Tissue Microarray Analysis: Stain patient tissue microarrays with FITC-conjugated KIF22 antibodies to correlate expression levels with clinical features and prognosis

• Co-localization Studies: Combine FITC-KIF22 staining with antibodies against MAPK-ERK pathway components to visualize their spatial relationships

• Fluorescence-based Protein Interaction Studies: Use FITC-KIF22 antibodies in proximity ligation assays to detect interactions with CDC25C and other cell cycle regulators

• Functional Analysis with KIF22 Modulation: Compare FITC-KIF22 staining patterns before and after siRNA knockdown to assess changes in cellular distribution and associated pathways

These approaches have revealed KIF22's association with poor prognosis in multiple myeloma patients, correlating with clinical features including LDH levels, β2-MG, and tumor cell percentage .

How can researchers use FITC-conjugated KIF22 antibodies to study its transcriptional regulatory functions?

To investigate KIF22's role in transcriptional regulation:

• Perform chromatin immunoprecipitation (ChIP) using FITC-conjugated KIF22 antibodies to identify DNA binding sites

• Combine with fluorescence microscopy to visualize KIF22's nuclear localization and potential co-localization with transcription factors

• Implement ChIP-seq analysis to identify genome-wide binding patterns of KIF22

• Correlate binding data with gene expression analysis after KIF22 knockdown/overexpression to determine direct transcriptional targets, such as CDC25C

This methodological approach has demonstrated that KIF22 directly regulates CDC25C by binding to its promoter, providing insight into how KIF22 influences cell cycle progression through transcriptional mechanisms in addition to its mechanical role during mitosis.

What are common issues when using FITC-conjugated KIF22 antibodies and how can they be resolved?

Incorporating KIF22 knockout cell lines as negative controls is particularly effective for troubleshooting, as demonstrated with HEK-293T KIF22 knockout cell lines showing complete loss of signal with anti-KIF22 antibodies .

How can researchers optimize FITC conjugation ratios for KIF22 antibodies?

Optimizing the FITC-to-antibody ratio is crucial for balancing signal strength against potential interference with antigen binding:

• Titration Approach: Prepare conjugates with varying FITC-to-antibody ratios (typically 2:1 to 20:1 molar ratios)

• Functional Testing: Test each conjugate for:

  • Signal intensity using flow cytometry or microscopy

  • Specificity by comparing staining patterns with unconjugated antibody controls

  • Affinity by comparing EC50 values in binding assays

• Spectral Analysis: Measure protein (280nm) and FITC (495nm) absorbance to calculate actual conjugation ratios

• Storage Stability: Evaluate signal preservation after storage at 4°C and -20°C for different time periods

The optimal ratio typically falls between 4-7 FITC molecules per antibody; higher ratios may increase fluorescence but can compromise antibody function through steric hindrance or altered charge distribution .

What strategies can address signal fading when using FITC-conjugated KIF22 antibodies in long-term imaging?

FITC is susceptible to photobleaching, which can limit extended imaging sessions. Researchers can implement these strategies:

• Anti-fade Reagents: Include anti-fade reagents containing radical scavengers in mounting media

• Oxygen Scavenging Systems: Incorporate enzymatic oxygen scavenging systems for live-cell imaging

• Reduced Exposure: Minimize excitation light intensity and exposure duration

• Alternative Conjugation: Consider more photostable fluorophores like Alexa Fluor 488 as alternatives to FITC

• Sequential Acquisition: Acquire FITC channel first in multi-color imaging experiments

• Image Processing: Apply computational approaches to correct for photobleaching in time-lapse experiments

These approaches help maintain signal integrity during experiments requiring extended or repeated imaging of KIF22 localization, particularly important for tracking dynamic changes during cell cycle progression.