ICK (Ab-159) Antibody

What is ICK (Ab-159) Antibody and what epitope does it recognize?

ICK (Ab-159) Antibody is a rabbit polyclonal antibody that specifically recognizes the region surrounding tyrosine 159 (T-D-Y(p)-V-S) in the human ICK (Intestinal Cell Kinase) protein. The antibody was generated using a synthesized non-phosphopeptide derived from this region. It has been affinity-purified from rabbit antiserum using epitope-specific immunogen chromatography, ensuring high specificity for the target sequence . This antibody detects endogenous levels of total ICK protein and is particularly useful for investigating ICK expression and function in cellular contexts.

What are the typical applications for ICK (Ab-159) Antibody?

ICK (Ab-159) Antibody has been validated for several research applications:

• Western Blotting (WB): Recommended dilution of 1:500-1:3000

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Cell-Based ELISA assays for monitoring ICK protein expression profiles in cultured cells

The antibody has demonstrated successful detection of endogenous ICK in various cell lines including 293 cells, cos-7 cells, and 3T3 cells, making it versatile for different experimental systems .

What species reactivity has been confirmed for this antibody?

ICK (Ab-159) Antibody has been experimentally confirmed to react with:

• Human samples

• Mouse samples

Western blot analyses have validated the antibody's reactivity across these species, making it suitable for comparative studies between human and mouse models .

What is the recommended protocol for using ICK (Ab-159) Antibody in Western blotting?

For optimal Western blotting results with ICK (Ab-159) Antibody:

• Prepare protein extracts from your cells of interest (successful results have been shown with 293, cos-7, and 3T3 cells)

• Resolve proteins by SDS-PAGE (the ICK protein has an expected molecular weight of approximately 65 kDa)

• Transfer proteins to a membrane using standard protocols

• Block the membrane using appropriate blocking buffer

• Dilute ICK (Ab-159) Antibody at 1:500-1:3000 in recommended antibody diluent

• Incubate membrane with diluted primary antibody (typically overnight at 4°C)

• Wash membrane thoroughly

• Incubate with appropriate HRP-conjugated secondary antibody

• Develop using your preferred detection method

The antibody has successfully detected ICK in various treatment conditions, including PBS-treated 3T3 cells (10μM, 60 minutes) .

How should ICK (Ab-159) Antibody be incorporated into a Cell-Based ELISA experiment?

For Cell-Based ELISA applications:

• Seed cells in a 96-well plate and culture to 80-90% confluence

• Fix cells with 4% paraformaldehyde (for adherent cells) or 8% paraformaldehyde (for suspension cells)

• For suspension cells, pre-coat wells with Poly-L-Lysine

• Permeabilize cells if targeting intracellular proteins

• Block with appropriate blocking buffer

• Prepare a 1:100 dilution of ICK (Ab-159) Antibody using Primary Antibody Diluent

• Add diluted primary antibody to wells and incubate (typically 2-3 hours at room temperature)

• Wash wells thoroughly

• Add HRP-conjugated secondary antibody and incubate

• Add Ready-to-Use Substrate and incubate for 30 minutes at room temperature in the dark with gentle shaking

• Add Stop Solution and read absorbance

Including GAPDH antibody (commonly provided in kits) as an internal control allows for normalization of results .

What storage conditions are optimal for maintaining ICK (Ab-159) Antibody activity?

For optimal performance and longevity:

• Store antibody at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles, which can degrade antibody performance

• The antibody is typically formulated in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, containing 150mM NaCl, 0.02% sodium azide, and 50% glycerol for stability

• Working aliquots can be prepared to minimize freeze-thaw cycles

• Expiration is typically 6 months from receipt when stored properly

What controls should be included when using ICK (Ab-159) Antibody in experimental applications?

To ensure experimental validity when using ICK (Ab-159) Antibody:

• Positive control: Use cell lines known to express ICK, such as 293 cells, cos-7 cells, or 3T3 cells

• Negative control: Include samples treated with synthesized peptide, which has been shown to block antibody binding

• Loading control: Include detection of housekeeping proteins such as GAPDH to normalize for loading differences

• Secondary antibody-only control: To assess non-specific binding

• Specificity validation: Consider using multiple antibodies targeting different epitopes of ICK to confirm results

Western blot analysis has shown that pre-incubation with the synthesized peptide effectively blocks antibody binding, confirming specificity .

How can ICK (Ab-159) Antibody be used in studying ICK's role in developmental processes?

ICK has been implicated in the development of multiple organ systems, particularly in cardiac development. To investigate its developmental roles:

• Temporal expression analysis: Use ICK (Ab-159) Antibody to track ICK expression across developmental stages in target tissues

• Co-localization studies: Combine with antibodies against developmental markers to establish spatial and temporal relationships

• Knockdown/knockout validation: Use the antibody to confirm successful reduction of ICK protein levels in genetic manipulation experiments

• Cell-based functional assays: Employ Cell-Based ELISA kits to quantify ICK expression changes during differentiation processes

• Phosphorylation analysis: Though this antibody targets total ICK, it could be used in conjunction with phospho-specific antibodies to study activity regulation

The antibody's specificity for the region around tyrosine 159 makes it particularly valuable for studying how this region may influence ICK function in developmental contexts .

What are the considerations for using ICK (Ab-159) Antibody in studies involving post-translational modifications?

When investigating post-translational modifications of ICK:

• Phosphorylation studies: While this antibody targets total ICK protein around Tyr159, it is not phospho-specific. For phosphorylation studies, consider using phospho-specific antibodies in parallel

• Treatment conditions: Compare ICK expression or modification across different treatment conditions, as demonstrated in studies with PBS treatment (10μM, 60 minutes)

• Protein-protein interactions: Consider using ICK (Ab-159) Antibody in co-immunoprecipitation studies to identify interaction partners

• Cellular localization changes: Use in immunofluorescence to track localization changes that may occur with post-translational modifications

• Degradation analysis: Monitor ICK protein levels in response to proteasome inhibitors or other modulators of protein degradation

Understanding the antibody's exact epitope (around Tyr159) is crucial when interpreting results related to modifications that might affect antibody binding .

What normalization methods are recommended when analyzing ICK expression using Cell-Based ELISA?

For robust quantification of ICK expression in Cell-Based ELISA experiments:

• GAPDH normalization: Use the included anti-GAPDH antibody as an internal positive control to normalize ICK absorbance values

• Crystal Violet whole-cell staining: Following colorimetric measurement, this method can determine cell density, allowing normalization to cell amounts to adjust for plating differences

• Total protein normalization: If detecting phosphorylated targets, normalization to total protein can provide insights into the proportion of modified protein

• Background subtraction: Always subtract values from secondary antibody-only controls to account for non-specific binding

• Standard curve calibration: If absolute quantification is needed, establish a standard curve using recombinant ICK protein

These approaches ensure that observed differences in ICK expression are not due to technical variations in cell number or protein loading .

How should contradictory results between ICK (Ab-159) Antibody and other ICK antibodies be interpreted?

When facing contradictory results between different ICK antibodies:

• Epitope differences: ICK (Ab-159) Antibody specifically targets the region around Tyr159. Other antibodies may target different epitopes that could be differentially accessible in certain experimental conditions

• Post-translational modifications: Modifications near the antibody's epitope might affect binding efficiency

• Antibody validation: Confirm antibody specificity using approaches such as peptide competition assays, as demonstrated in Western blots where synthesized peptide blocked binding

• Experimental conditions: Different lysis buffers, fixation methods, or blocking agents can affect epitope accessibility

• Cross-reactivity: Assess whether contradictory results might stem from cross-reactivity with related proteins

For comprehensive studies, using multiple antibodies targeting different regions of ICK can provide a more complete understanding of the protein's expression and function .

What are the key limitations to consider when interpreting results obtained with ICK (Ab-159) Antibody?

Important limitations to consider include:

• Polyclonal nature: As a polyclonal antibody, batch-to-batch variation may occur, potentially affecting consistency across experiments

• Cross-reactivity potential: While validated for human and mouse samples, potential cross-reactivity with other species or proteins should be considered

• Epitope accessibility: Certain experimental conditions may affect the accessibility of the epitope around Tyr159

• Non-phospho-specific: This antibody detects total ICK regardless of phosphorylation status at Tyr159

• Detection methods: Sensitivity may vary between different detection systems (chemiluminescence, fluorescence, colorimetric)

Understanding these limitations is crucial for appropriate experimental design and data interpretation .

How does the specificity of ICK (Ab-159) Antibody compare to other commercially available ICK antibodies?

When evaluating ICK (Ab-159) Antibody against other commercial options:

• Epitope targeting: ICK (Ab-159) targets specifically the region around Tyr159, whereas other antibodies may target N-terminal, C-terminal, or internal regions

• Validation methods: This antibody has been validated by Western blotting in multiple cell lines and with peptide competition assays

• Species reactivity: Confirmed for human and mouse samples, which may differ from other antibodies' reactivity profiles

• Application range: Validated for Western blot and ELISA applications, while other antibodies may be validated for additional applications like IHC or IP

• Phospho-specificity: Unlike phospho-specific antibodies, ICK (Ab-159) detects total ICK protein

How is ICK (Ab-159) Antibody being utilized in current research on ICK's role in cellular signaling pathways?

Current research applications include:

• Kinase activity studies: Monitoring ICK expression in response to various stimuli to understand its regulation

• Developmental biology: Investigating ICK's reported role in cardiac development and other organ systems

• Signal transduction: Examining ICK's position within cellular signaling cascades

• Cell cycle regulation: Studying potential roles in cell proliferation and division

• Comparative expression analysis: Using the antibody's cross-reactivity with human and mouse samples to conduct comparative studies

The antibody's specificity makes it valuable for distinguishing ICK from other related kinases in these research contexts .

What methodological advances have improved the application of antibodies like ICK (Ab-159) in protein characterization?

Recent methodological advances include:

• Cell-Based ELISA technologies: Allowing for quantification of ICK expression in intact cells without the need for cell lysis

• Multiplexed detection systems: Enabling simultaneous detection of ICK alongside other proteins of interest

• Automated Western blotting platforms: Improving reproducibility and quantification of ICK detection

• Super-resolution microscopy: Enhancing the spatial resolution of ICK localization studies when using the antibody for immunofluorescence

• Reverse-Phase Protein Arrays (RPPA): Allowing high-throughput analysis of ICK across multiple samples simultaneously

These technological advances have expanded the utility and reliability of antibody-based detection methods for ICK research .

What documentation should be maintained when using ICK (Ab-159) Antibody in published research?

For research integrity and reproducibility, maintain:

• Antibody information: Full product details including catalog number, lot number, manufacturer, and storage conditions

• Experimental protocols: Detailed methods including dilutions, incubation times, and detection systems

• Validation data: Evidence of antibody specificity such as peptide competition assays or knockdown controls

• Positive and negative controls: Documentation of all controls used to validate results

• Image acquisition parameters: For Western blots or immunofluorescence, record exposure times, gain settings, and any image processing steps

This documentation is essential for research reproducibility and compliance with journal requirements for antibody-based research .

What validation approaches should be employed when first using ICK (Ab-159) Antibody in a new experimental system?

When introducing this antibody to a new experimental system:

• Preliminary titration: Test multiple dilutions to determine optimal concentration for your specific application

• Peptide competition: Confirm specificity using the synthesized peptide (T-D-Y-V-S) as a competitive inhibitor

• Known positive samples: Include samples with confirmed ICK expression (e.g., 293 cells, cos-7 cells)

• Knockdown verification: If possible, include ICK knockdown or knockout samples to confirm signal specificity

• Cross-platform validation: Verify results using complementary techniques (e.g., if using for Western blot, confirm with qPCR)