KIF22 Antibody, HRP conjugated

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

KIF22, also known as Kid or kinesin-like DNA-binding protein, belongs to the kinesin superfamily of motor proteins. It plays critical roles in spindle formation and chromosome movement during mitosis and meiosis by binding to both microtubules and DNA . KIF22 has particular significance in cell division research as it contributes to the congression of laterally attached chromosomes in NDC80-depleted cells . Studies of KIF22 can provide insights into fundamental cellular processes and potential dysregulation in pathological conditions, making it an important target for antibody-based detection in research applications focused on cell cycle regulation, chromosome dynamics, and related cellular mechanisms.

How do researchers distinguish between polyclonal and monoclonal KIF22 antibodies?

Polyclonal KIF22 antibodies, such as those generated in rabbits (e.g., ab222187), recognize multiple epitopes across the KIF22 protein, particularly in regions like amino acids 550-650 . These antibodies offer advantages in sensitivity due to multi-epitope recognition but may show batch-to-batch variation. In contrast, monoclonal KIF22 antibodies like the rabbit monoclonal EP2748 (ab284779) target a single specific epitope, ensuring consistent recognition across experiments with higher specificity but potentially lower signal strength . The selection between these types should be based on experimental goals: polyclonal antibodies are often preferred for initial detection and applications requiring high sensitivity, while monoclonal antibodies are advantageous for reproducible experiments requiring consistent epitope targeting across multiple studies.

What validated applications are supported by available KIF22 antibodies?

KIF22 antibodies have been validated across multiple applications with specific reactivity profiles as demonstrated in the literature and manufacturer testing. Western blot applications typically use dilutions ranging from 1:500-1:2000, with observed molecular weights between 68-73 kDa . Immunohistochemistry applications use dilutions of 1:250-1:1000, with validated reactivity in human tissues including stomach cancer, breast cancer, and cervical cancer tissues . Immunofluorescence typically employs concentrations of 0.25-2 μg/mL (for ICC-IF) . Immunoprecipitation protocols recommend using 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate . These applications have been tested across human, mouse, and rat samples with confirmed reactivity .

What are the optimized protocols for Western blot detection of KIF22?

For optimal Western blot detection of KIF22, researchers should follow this methodological approach: Begin with protein extraction using RIPA buffer supplemented with protease inhibitors. Load 20-40 μg of total protein per lane on an 8-10% SDS-PAGE gel (KIF22 has an observed molecular weight of 68-73 kDa) . After transfer to a PVDF membrane (wet transfer recommended at 100V for 90 minutes), block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature. Incubate with primary KIF22 antibody at 0.04-0.4 μg/mL or at dilutions between 1:500-1:2000 in blocking buffer overnight at 4°C . Following primary antibody incubation, wash the membrane thoroughly with TBST (3 × 10 minutes) and incubate with HRP-conjugated secondary antibody at 1:50 dilution for 1 hour at room temperature . Develop using ECL substrate and expose to film or digital imager. For enhanced specificity, include validated positive controls such as Jurkat cells, human heart tissue, MKN-45 cells, or T-47D cells, which have been confirmed to express KIF22 .

How should researchers optimize HRP signal development when using KIF22 antibodies?

Signal development optimization with HRP-conjugated detection systems for KIF22 antibodies requires careful attention to several methodological factors. First, substrate selection should match the expected expression level of KIF22 in your samples—standard ECL substrates are suitable for normal expression levels, while enhanced chemiluminescent substrates (femto-level sensitivity) should be used for low expression samples. The incubation time with substrate requires empirical determination, typically starting with 1-5 minutes. For quantitative analysis, researchers should establish a linear dynamic range by performing serial dilutions of positive control samples (such as Jurkat cells) . If using HRP-conjugated secondary antibodies, maintain the recommended 1:50 dilution for optimal signal-to-noise ratio . To minimize background, add 0.05-0.1% Tween-20 to antibody dilution buffers and ensure thorough washing steps (at least 3 × 10 minutes) between antibody incubations. For challenging samples, consider signal enhancement systems like tyramide signal amplification, which can provide up to 100-fold signal enhancement while maintaining specificity.

What troubleshooting approaches address common issues with KIF22 immunodetection?

When encountering challenges with KIF22 immunodetection, systematic troubleshooting approaches should be implemented. For weak or absent signals, consider: (1) increasing antibody concentration incrementally, (2) extending incubation times (up to 16 hours at 4°C), (3) optimizing antigen retrieval for IHC applications (the KIF22 antibody works best with TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative) , or (4) switching to a more sensitive detection system. For high background issues: (1) test more stringent blocking (5% BSA instead of milk), (2) increase washing duration and frequency, (3) further dilute antibodies, or (4) pre-absorb the antibody with non-specific proteins. When multiple bands appear in Western blot, validate using KIF22 knockout/knockdown samples as negative controls, as demonstrated with the HEK-293T KIF22 knockout cell line . For inconsistent results between experiments, standardize protein loading (20-40 μg), use fresh antibody aliquots (avoiding freeze-thaw cycles), and include consistent positive controls in each experiment.

How can researchers optimize KIF22 antibodies for studying mitotic spindle dynamics?

For studying mitotic spindle dynamics using KIF22 antibodies, implement a multi-modal imaging approach combining fixed and live-cell techniques. For fixed-cell immunofluorescence, use PFA fixation (4% for 15 minutes) followed by Triton X-100 permeabilization (0.2% for 10 minutes) as validated with MCF7 cells . Use KIF22 antibody at 4 μg/ml for optimal spindle visualization . For co-localization studies, combine KIF22 antibody with markers for kinetochores (CENP-A), microtubules (α-tubulin), and chromosomes (DAPI). The most informative mitotic phases for KIF22 dynamics are prometaphase and metaphase. For super-resolution microscopy, structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy provides the optimal resolution (80-120 nm) required for detailed spindle structure analysis. When designing live-cell experiments, consider performing initial validation with fixed-cell KIF22 immunofluorescence to confirm antibody specificity and optimal dilution before proceeding to more complex dynamic studies. Time-lapse imaging intervals of 1-3 minutes are recommended to capture KIF22 movement along the spindle without significant photobleaching.

What methods ensure antibody specificity validation for KIF22 research?

Comprehensive validation of KIF22 antibody specificity requires a multi-layered approach combining molecular, cellular, and analytical techniques. Begin with knockout validation using KIF22 knockout cell lines, such as the HEK-293T KIF22 knockout cell line that has been successfully used to validate anti-KIF22 antibody [EP2748] . Implement RNAi-mediated knockdown as a complementary approach, comparing KIF22 signal reduction between siRNA-treated and control samples across multiple time points (24-72 hours post-transfection). Peptide competition assays provide additional validation by pre-incubating the antibody with excess immunizing peptide; specific signals should be significantly reduced or eliminated. Cross-reactivity assessment should include testing the antibody against a protein array containing 364 human recombinant protein fragments, as is done for Prestige Antibodies . For quantitative validation, perform dose-response experiments using recombinant KIF22 protein standards to establish antibody sensitivity and linear detection range. Documentation of antibody validation should include images from multiple applications (WB, IF, IHC) with appropriate controls, showing expected localization patterns (nuclear during interphase, spindle-associated during mitosis) and molecular weight (68-73 kDa).

What are the optimal methods for studying KIF22 interactions using co-immunoprecipitation?

For studying KIF22 protein interactions via co-immunoprecipitation (co-IP), implement a carefully optimized protocol tailored to preserve KIF22's functional interactions. Begin with gentle cell lysis using a non-denaturing buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol) supplemented with protease and phosphatase inhibitors. Pre-clear lysates containing 1.0-3.0 mg total protein with protein A/G beads for 1 hour at 4°C. For the IP, use 0.5-4.0 μg of KIF22 antibody per reaction , incubating overnight at 4°C with gentle rotation. When selecting antibody clones for co-IP, polyclonal antibodies often perform better than monoclonal antibodies due to their recognition of multiple epitopes, increasing capture efficiency. Follow with addition of protein A/G beads for 2-4 hours at 4°C. Wash beads 4-5 times with lysis buffer containing reduced detergent (0.1% NP-40). Elute bound proteins with 2X SDS sample buffer at 95°C for 5 minutes. For downstream analysis of interacting partners, consider mass spectrometry or targeted Western blot for suspected interaction partners such as chromosome components, other kinesins, or cell cycle regulators. Key controls should include: (1) IgG-matched negative control, (2) input samples (5-10% of starting material), (3) verification of successful KIF22 pull-down in a separate blot, and (4) reverse co-IP validation for key interaction partners.

How should researchers optimize chromogenic versus chemiluminescent detection with HRP-conjugated systems for KIF22?

The selection and optimization between chromogenic and chemiluminescent detection systems for KIF22 visualization depends on research objectives and required sensitivity. Chemiluminescent detection offers superior sensitivity, typically detecting KIF22 at concentrations 10-100 fold lower than chromogenic methods. For Western blot applications, standard ECL substrates provide detection limits in the high picogram range, while enhanced ECL systems can extend sensitivity to the low picogram range. Optimal exposure times typically range from 30 seconds to 5 minutes, with longer exposures risking background development. For chromogenic detection in IHC applications, DAB (3,3'-diaminobenzidine) substrate is most commonly used with HRP-conjugated systems. Optimization requires careful timing of the substrate development (typically 1-10 minutes) with monitoring under a microscope to achieve optimal signal-to-noise ratio. The key advantage of chromogenic detection is the generation of permanent preparations that do not fade over time, while chemiluminescent signals are transient. When quantitative analysis is required, chemiluminescent detection provides superior linear dynamic range (typically 2-3 orders of magnitude) compared to chromogenic methods. For multiplex detection of KIF22 alongside other proteins, chromogenic systems using different substrates (DAB, AEC, etc.) allow visualization of multiple targets in distinct colors on the same tissue section.

What storage and handling practices maximize the shelf-life and performance of HRP-conjugated antibody systems?

To maximize shelf-life and maintain optimal activity of HRP-conjugated antibody systems, implement these evidence-based handling and storage practices: Store stock antibody solutions at -20°C in small single-use aliquots to minimize freeze-thaw cycles, as each cycle can reduce activity by 10-20% . For working dilutions, prepare fresh on the day of use and store at 4°C for no more than 24 hours. Incorporate stabilizing proteins such as BSA (0.1-1%) into storage buffers to prevent non-specific adsorption to tube walls and maintain antibody activity . Avoid repeated freeze-thaw cycles by creating multiple small-volume aliquots (10-20 μL) upon receipt of the antibody. Protect HRP-conjugated antibodies from light exposure, as light can accelerate oxidation of the heme group in HRP, leading to decreased enzymatic activity. When handling, avoid introducing bacterial contamination by using sterile technique when accessing antibody stocks. For long-term storage beyond 6 months, adding glycerol to a final concentration of 50% helps prevent freeze-thaw damage and maintains antibody stability . Before each use, centrifuge antibody vials briefly to collect all liquid at the bottom of the tube. Under optimal storage conditions, HRP-conjugated antibody systems typically maintain >80% activity for 12 months at -20°C.

What controls are essential for validating KIF22 antibody specificity in experimental protocols?

A comprehensive control strategy for KIF22 antibody experiments should include multiple validation approaches to ensure reliable data interpretation. Primary antibody controls should include: (1) KIF22 knockout or knockdown samples, such as HEK-293T KIF22 knockout cell line, which has been validated with anti-KIF22 antibody [EP2748] ; (2) No-primary-antibody controls to establish background staining levels; (3) Isotype-matched controls (rabbit IgG for rabbit anti-KIF22) to identify non-specific binding; and (4) Peptide competition controls using the immunizing peptide to confirm epitope specificity. Sample-specific positive controls should include tissues or cell lines with confirmed KIF22 expression, such as Jurkat cells, human heart tissue, MKN-45 cells, or T-47D cells . For immunohistochemistry applications, include known positive tissues such as human stomach cancer, breast cancer, or cervical cancer tissue . For technical validation, include loading controls appropriate to the application: GAPDH (36 kDa) for Western blot and reference markers for subcellular compartments in immunofluorescence. Quantitative controls should include standard curves using recombinant KIF22 protein to establish the linear detection range of the assay and enable accurate quantification.

How can researchers design experiments to study KIF22 across different cell cycle phases?

Studying KIF22 across different cell cycle phases requires thoughtful experimental design integrating synchronization techniques, imaging approaches, and quantitative analysis. Begin with cell synchronization using one of these validated methods: (1) Double thymidine block for G1/S boundary arrest; (2) Nocodazole (100 ng/mL, 16h) for metaphase arrest; or (3) RO-3306 (10 μM, 16h) for G2 arrest. For each method, validate synchronization efficiency using flow cytometry with propidium iodide staining before proceeding to KIF22 analysis. After synchronization release, collect cells at defined timepoints (e.g., 0h, 2h, 4h, 6h, 8h, 10h) to capture different cell cycle phases. For immunofluorescence analysis, co-stain with KIF22 antibody (4 μg/ml) and cell cycle phase markers: Cyclin D1 (G1), PCNA (S-phase), phospho-histone H3 (M-phase). Use confocal microscopy with z-stacking (0.3-0.5 μm intervals) to capture the full 3D distribution of KIF22 throughout the cell. For quantitative assessment, measure KIF22 protein levels by Western blot throughout the cell cycle, normalizing to total protein content rather than housekeeping proteins (which may fluctuate during the cell cycle). Additionally, assess KIF22 post-translational modifications using phosphorylation-specific antibodies if available, as these often regulate kinesin activity in a cell cycle-dependent manner.

What approaches resolve contradictory data when using different KIF22 antibody clones?

When faced with contradictory results from different KIF22 antibody clones, implement a systematic resolution strategy incorporating multiple validation techniques and detailed protocol analysis. First, perform epitope mapping to determine the binding regions of each antibody clone. Monoclonal antibodies like EP2748 and polyclonal antibodies recognizing different regions (e.g., aa 550-650) may yield different results if the epitopes undergo post-translational modifications or are masked in certain protein complexes or conformations. Conduct parallel validation experiments using multiple detection methods (Western blot, immunoprecipitation, immunofluorescence) with each antibody clone under identical conditions. Use knockout validation as the gold standard by testing all antibody clones against KIF22 knockout cell lines to identify truly specific signals. Cross-reference with orthogonal detection methods such as mass spectrometry or RNA expression data to confirm KIF22 presence and quantity. For contradictory subcellular localization data, perform subcellular fractionation followed by Western blot to biochemically verify the distribution pattern. Consider fixation method artifacts, particularly for KIF22, as different fixatives (PFA versus methanol) can yield different accessibility to epitopes. Document all experimental conditions in detail, including antibody lot numbers, dilutions, incubation times, buffer compositions, and sample preparation methods, as these variables can significantly impact results. If contradictions persist after thorough validation, consider the possibility that both antibodies are detecting KIF22 correctly but under different conditions or conformational states.