ICK Antibody, Biotin conjugated

What is ICK Antibody, Biotin conjugated and what cellular processes does it target?

ICK Antibody, Biotin conjugated is a polyclonal antibody raised against Serine/threonine-protein kinase ICK (Intestinal cell kinase). The target protein plays critical roles in ciliogenesis and regulates intraflagellar transport (IFT) speed in a cAMP and mTORC1 signaling-dependent manner. ICK also negatively regulates cilium length, with its kinase activity being essential for this function. Additionally, ICK is involved in the development of multiple organ systems, particularly cardiac development, and regulates the ciliary localization of SHH (Sonic Hedgehog) pathway components and IFT components at ciliary tips .

The biotin conjugation refers to the covalent attachment of biotin molecules to the antibody, which enables detection through secondary reagents like streptavidin or avidin conjugated to enzymes, fluorophores, or other detection molecules. This conjugation leverages the extremely high affinity between biotin and streptavidin/avidin, providing enhanced sensitivity in various immunoassay applications .

What are the technical specifications of commercially available ICK Antibody, Biotin conjugated?

Based on multiple commercial sources, typical specifications for ICK Antibody, Biotin conjugated include:

This antibody recognizes the human ICK protein, which has aliases including Intestinal cell kinase (hICK), Laryngeal cancer kinase 2 (LCK2), and MAK-related kinase (MRK) .

How does the biotin conjugation affect antibody performance compared to unconjugated alternatives?

Biotin conjugation provides several methodological advantages over unconjugated antibodies while introducing certain constraints researchers should consider:

The biotin-streptavidin detection system offers amplified signal due to the multiple biotin binding sites on each streptavidin molecule (four binding sites per molecule). This amplification makes biotin-conjugated antibodies particularly valuable for detecting proteins expressed at low levels, which might be below the detection threshold of direct detection methods .

Additionally, researchers should be aware that endogenous biotin in biological samples can contribute to background signal in certain applications, particularly when working with biotin-rich tissues like liver, kidney, or brain. In such cases, biotin-blocking steps must be incorporated into experimental protocols to ensure specificity .

What are the optimal conditions for using ICK Antibody, Biotin conjugated in ELISA applications?

When designing ELISA experiments with ICK Antibody, Biotin conjugated, researchers should implement the following methodological considerations for optimal results:

Protocol Optimization:

• Sample preparation: Samples should be prepared in a buffer compatible with the antibody's storage buffer (typically PBS-based). For cell lysates, a buffer containing 0.01M PBS (pH 7.4) with mild detergents like 0.1% Triton X-100 is recommended.

• Antibody dilution: Initial testing should include a titration series (typically 1:500, 1:1000, 1:2000, and 1:5000) to determine the optimal antibody concentration that provides the best signal-to-noise ratio. For ICK Antibody, Biotin conjugated, a starting dilution of 1:1000 is often appropriate .

• Detection system: Use high-quality streptavidin-conjugated enzymes (HRP or AP) for detection. The streptavidin reagent should be titrated independently (typical range: 1:5000 to 1:20000).

• Incubation conditions: Optimal binding occurs at room temperature (22-25°C) for 1-2 hours or overnight at 4°C. Shorter incubations may be possible at higher antibody concentrations but may increase background.

• Washing steps: Implement at least 3-5 washes with PBS containing 0.05% Tween-20 between each step to minimize non-specific binding and background signal.

These guidelines should be adjusted based on experimental requirements and initial validation results. Since the antibody has been specifically validated for ELISA applications, researchers can expect consistent performance when following these methodological approaches .

How should researchers validate the specificity of ICK Antibody, Biotin conjugated in their experimental system?

Comprehensive validation of ICK Antibody, Biotin conjugated specificity requires a multi-faceted approach:

Validation Strategy:

• Positive and negative controls: Include samples with known ICK expression levels. Human cell lines with documented ICK expression (e.g., certain intestinal epithelial cell lines) serve as positive controls. Cell lines known to express minimal ICK or cells where ICK has been knocked down via siRNA/CRISPR can serve as negative controls.

• Peptide competition assay: Pre-incubate the antibody with excess recombinant ICK protein (specifically the immunogen fragment, amino acids 150-275) before application to samples. This should abolish specific signal if the antibody is truly specific to ICK.

• Western blot correlation: Although this antibody is primarily validated for ELISA, running parallel Western blot experiments with a validated non-biotin conjugated ICK antibody can provide confirmation of specificity by demonstrating detection of the expected 73 kDa band corresponding to the ICK protein.

• Cross-reactivity assessment: As this antibody is raised against human ICK, test for potential cross-reactivity if working with non-human samples. The immunogen sequence (amino acids 150-275) should be compared across species to predict potential cross-reactivity.

• Blocking endogenous biotin: When working with biotin-rich samples, incorporate avidin/streptavidin blocking steps followed by biotin blocking before adding the biotin-conjugated antibody to minimize false positive signals from endogenous biotin.

Documentation of these validation steps substantially strengthens the reliability of research findings and should be included in materials and methods sections of publications .

What are the recommended storage and handling protocols to maintain ICK Antibody, Biotin conjugated activity?

Proper storage and handling of ICK Antibody, Biotin conjugated is critical for maintaining its activity and extending its usable lifetime:

Optimal Storage and Handling Protocol:

• Initial handling: Upon receipt, the antibody should be aliquoted into single-use volumes (typically 10-20 μL) in sterile microcentrifuge tubes to minimize freeze-thaw cycles.

• Storage temperature: Store aliquots at -20°C or preferably -80°C for long-term stability. Avoid storing at 4°C for more than 1-2 weeks.

• Freeze-thaw management: Limit freeze-thaw cycles to a maximum of 5, as repeated freezing and thawing can degrade both the antibody protein and the biotin conjugate. Record the number of freeze-thaw cycles on each tube.

• Protection from light: Biotin conjugates are sensitive to light exposure. Store in amber tubes or wrap in aluminum foil to protect from light, particularly during extended storage periods.

• Working solution preparation: When preparing working dilutions, use high-quality, sterile-filtered buffers. Once diluted, the antibody should ideally be used within the same day. If necessary, diluted antibody can be stored at 4°C for up to 24 hours but should be protected from light.

• Microbial contamination prevention: Use sterile technique when handling the antibody to prevent microbial growth, even though the storage buffer contains the preservative Proclin 300.

Adherence to these storage and handling protocols will help maintain antibody activity and ensure consistent experimental results. The manufacturer's recommendations should always take precedence if they differ from these general guidelines .

How can researchers adapt ICK Antibody, Biotin conjugated for use in multiplexed immunoassays?

Multiplexed immunoassays with ICK Antibody, Biotin conjugated require strategic planning to overcome potential limitations of biotin-streptavidin systems in multi-target detection:

Multiplexing Methodology:

• Sequential detection approach: When combining with other biotin-conjugated antibodies, implement sequential rather than simultaneous detection. This involves:

  • Complete primary detection of ICK using the biotin-conjugated antibody

  • Signal development and thorough washing

  • Blocking of remaining biotin binding sites using excess free biotin

  • Proceed with the next biotin-conjugated antibody

• Streptavidin-fluorophore selection: For fluorescence-based multiplexing, select streptavidin conjugated to spectrally distinct fluorophores that can be clearly differentiated by your detection system. Common combinations include:

  • Streptavidin-Cy3 (emission ~570 nm)

  • Streptavidin-Cy5 (emission ~670 nm)

  • Streptavidin-FITC (emission ~520 nm)

• Alternative reporter systems: Consider using one biotin-conjugated antibody (such as ICK) with streptavidin-HRP, while using directly labeled primary or secondary antibodies for other targets.

• Signal separation strategies: For chromogenic assays, utilize:

  • Different substrates (TMB, DAB, AEC) for distinct color development

  • Different enzymes (HRP vs. AP) with their specific substrates

  • Sequential development and imaging before proceeding to the next target

• Validation controls: Include single-antibody controls alongside multiplexed samples to verify that detection of each target is not compromised in the multiplexed format.

These approaches enable researchers to effectively include ICK Antibody, Biotin conjugated in multiplexed analyses while maintaining specificity and sensitivity for each target .

What strategies can researchers employ when facing high background or non-specific binding issues with ICK Antibody, Biotin conjugated?

High background or non-specific binding with biotin-conjugated antibodies like ICK Antibody requires systematic troubleshooting approaches:

Background Reduction Protocol:

• Endogenous biotin blocking:

  • Implement a pre-blocking step with unconjugated avidin/streptavidin (10-50 μg/mL for 15-30 minutes)

  • Follow with excess free biotin (50-200 μg/mL for 15-30 minutes)

  • This sequential blocking saturates endogenous biotin and fills remaining binding sites on avidin/streptavidin

• Optimize antibody concentration:

  • Create a dilution series (1:500 to 1:5000) to identify the minimal concentration that maintains specific signal

  • Higher antibody concentrations often increase non-specific binding

• Blocking optimization:

  • Test different blocking agents (BSA, casein, commercial blocking buffers)

  • Extend blocking time to 2 hours at room temperature or overnight at 4°C

  • Include 0.1-0.5% Tween-20 in blocking buffer to reduce hydrophobic interactions

• Buffer modifications:

  • Add 0.1-0.5 M NaCl to reduce ionic interactions

  • Include 1-5% normal serum from the same species as the sample

  • Add 0.1% gelatin or 0.5% nonfat dry milk as additional blocking agents

• Enhanced washing protocol:

  • Increase wash buffer stringency (0.1% instead of 0.05% Tween-20)

  • Perform additional wash steps (5-7 instead of standard 3)

  • Extend wash duration (5 minutes per wash with gentle agitation)

• Sample pre-treatment:

  • Pre-absorb samples with Protein G beads to remove potentially interfering components

  • Filter samples through 0.45 μm filters to remove aggregates

Systematic implementation and documentation of these approaches can significantly improve signal-to-noise ratios in experimental applications .

How can researchers quantitatively assess the binding kinetics and affinity of ICK Antibody, Biotin conjugated?

Quantitative assessment of binding kinetics and affinity for ICK Antibody, Biotin conjugated can be achieved through several complementary biophysical methods:

Kinetics and Affinity Determination Methodology:

• Surface Plasmon Resonance (SPR):

  • Immobilize recombinant ICK protein on a sensor chip

  • Flow the biotin-conjugated antibody at various concentrations over the surface

  • Measure association (ka) and dissociation (kd) rate constants

  • Calculate the equilibrium dissociation constant (KD = kd/ka)

  • Expected high-affinity antibodies should exhibit KD values in the nanomolar to picomolar range

• Bio-Layer Interferometry (BLI):

  • Load streptavidin-coated biosensors with the biotin-conjugated antibody

  • Expose to varying concentrations of recombinant ICK protein

  • Monitor real-time binding curves to determine association and dissociation kinetics

  • This method is particularly suitable for biotin-conjugated antibodies due to the straightforward immobilization

• Competitive ELISA for Relative Affinity Assessment:

  • Coat plates with recombinant ICK protein

  • Pre-incubate biotin-conjugated antibody with varying concentrations of soluble ICK protein

  • Apply the mixture to the coated plate

  • Detect bound antibody with streptavidin-HRP

  • Plot inhibition curves and calculate IC50 values (lower IC50 indicates higher affinity)

• Isothermal Titration Calorimetry (ITC):

  • Directly measure thermodynamic parameters of binding

  • Provides enthalpy (ΔH), entropy (ΔS), and Gibbs free energy (ΔG) of binding

  • Calculate the number of binding sites and binding constants

  • Requires larger amounts of both antibody and antigen

These quantitative approaches provide crucial information for optimizing experimental conditions and interpreting results, particularly in applications requiring precise understanding of antibody-antigen interaction dynamics .

How can ICK Antibody, Biotin conjugated be utilized in studies of ciliopathies and related developmental disorders?

ICK plays a crucial role in ciliogenesis and ciliary function, making ICK Antibody, Biotin conjugated a valuable tool for investigating ciliopathies and developmental disorders:

Methodological Approaches for Ciliopathy Research:

• Co-localization studies in ciliated cells:

  • Use ICK Antibody, Biotin conjugated in combination with streptavidin-fluorophore conjugates

  • Counter-stain with established ciliary markers (acetylated α-tubulin, ARL13B)

  • Analyze co-localization patterns using confocal microscopy

  • Quantify ICK distribution along the ciliary axoneme in normal vs. disease models

• Functional analysis of ICK phosphorylation targets:

  • ICK phosphorylates KIF3A and other ciliary proteins

  • Use ICK Antibody, Biotin conjugated in proximity ligation assays (PLA) with antibodies against potential substrates

  • Implement phosphorylation-specific assays to correlate ICK localization with substrate modification

• Developmental expression profiling:

  • Analyze ICK expression during different developmental stages

  • Compare expression patterns in normal development vs. models of ECO (endocrine-cerebro-osteodysplasia) syndrome and other ICK-related disorders

  • Correlate ICK expression with phenotypic manifestations of ciliopathies

• Screening assays for therapeutic modulators:

  • Develop high-throughput ELISA-based screens using ICK Antibody, Biotin conjugated

  • Test compounds that potentially restore normal ICK activity or localization

  • Quantify changes in ICK expression or localization in response to therapeutic candidates

• Genetic interaction studies:

  • Combine with other ciliopathy protein markers in models with genetic modifications

  • Assess how alterations in SHH pathway components affect ICK localization

  • Investigate ICK relationships with IFT components at ciliary tips

These approaches can provide insights into the molecular mechanisms of ciliopathies and potentially identify therapeutic targets for developmental disorders associated with ciliary dysfunction .

What are the considerations for applying ICK Antibody, Biotin conjugated in single-cell analysis techniques?

Applying ICK Antibody, Biotin conjugated to single-cell analysis requires special considerations to maintain sensitivity and specificity at the individual cell level:

Single-Cell Analysis Implementation:

• Single-cell cytometry protocols:

  • Optimize cell fixation (4% paraformaldehyde, 10 minutes) and permeabilization (0.1% Triton X-100, 5 minutes)

  • Implement sequential staining: primary targets first, then ICK Antibody, Biotin conjugated

  • Use streptavidin-conjugated fluorophores with brightness appropriate for the expected ICK expression level

  • Include viability dyes to exclude dead cells which often show non-specific antibody binding

• Signal amplification methods:

  • Employ tyramide signal amplification (TSA) with streptavidin-HRP

  • Consider proximity ligation assay (PLA) for detecting ICK interactions with other proteins

  • Use branched DNA techniques for correlating ICK protein with mRNA levels

• Microfluidic applications:

  • Develop on-chip immunoassays with immobilized capture antibodies against ICK

  • Detect using biotin-conjugated antibody with streptavidin-fluorophore

  • Calibrate detection systems with recombinant ICK protein standards

• Mass cytometry considerations:

  • For CyTOF applications, use a metal-tagged streptavidin (e.g., streptavidin-Gd) to detect biotin-conjugated ICK antibody

  • Include barcoding strategies to minimize batch effects across samples

  • Implement careful compensation when multiplexing with other metal-tagged antibodies

• Single-cell validation approaches:

  • Correlate antibody staining with mRNA expression in the same cell

  • Use imaging to confirm subcellular localization predicted by ICK's known functions

  • Include multiple antibodies targeting different epitopes of ICK to confirm specificity

These methodological considerations enable researchers to effectively incorporate ICK Antibody, Biotin conjugated into emerging single-cell analysis platforms while maintaining data quality and reliability .

How can researchers leverage ICK Antibody, Biotin conjugated for investigating interactions between cilia function and Sonic Hedgehog (SHH) signaling?

The role of ICK in regulating ciliary localization of SHH pathway components makes ICK Antibody, Biotin conjugated particularly valuable for investigating this signaling pathway:

SHH-Cilia Interaction Analysis Protocol:

• Co-immunoprecipitation strategies:

  • Use streptavidin-coated magnetic beads to capture ICK Antibody, Biotin conjugated

  • Pull down ICK and associated proteins from ciliated cell lysates

  • Analyze precipitates for SHH pathway components (PTCH1, SMO, GLI proteins)

  • Quantify interaction strength under different signaling conditions (Hedgehog stimulation vs. inhibition)

• Ciliary trafficking analysis:

  • Implement live-cell imaging using indirect detection of ICK Antibody, Biotin conjugated

  • Track movement of ICK in relation to fluorescently tagged SHH components

  • Analyze trafficking kinetics using kymograph analysis

  • Correlate ICK localization with SHH pathway activation

• Proximity-based interaction studies:

  • Utilize proximity ligation assays (PLA) between ICK and SHH pathway components

  • Implement FRET analysis using streptavidin-fluorophores paired with directly labeled SHH components

  • Quantify spatial relationships at nanometer resolution using super-resolution microscopy

• Functional modulation approach:

  • Combine ICK inhibition (small molecules or genetic manipulation) with SHH pathway activation/inhibition

  • Monitor changes in ciliary localization using ICK Antibody, Biotin conjugated

  • Correlate localization patterns with downstream SHH target gene expression

  • Develop quantitative models of feedback relationships

• Developmental context analysis:

  • Examine temporal changes in ICK-SHH relationships during development

  • Compare normal embryonic patterning with models of ciliopathies

  • Correlate changes in ICK localization with developmental phenotypes

These methodological approaches enable researchers to unravel the complex relationships between cilia function, ICK activity, and SHH signaling, potentially leading to new therapeutic targets for developmental disorders and cancers associated with SHH pathway dysregulation .

What statistical approaches are recommended for quantifying ICK expression levels detected using biotin-conjugated antibodies?

Quantitative analysis of ICK expression requires appropriate statistical methods to ensure reliable interpretation of data obtained with biotin-conjugated antibodies:

Statistical Analysis Framework:

These statistical approaches ensure rigorous quantification of ICK expression levels and facilitate meaningful comparisons across experimental conditions, cell types, or disease states .

How should researchers evaluate potential cross-reactivity or non-specific binding of ICK Antibody, Biotin conjugated in complex biological samples?

Systematic evaluation of cross-reactivity and non-specific binding is essential for accurate interpretation of results with ICK Antibody, Biotin conjugated:

Cross-Reactivity Assessment Protocol:

• In silico analysis:

  • Compare the immunogen sequence (amino acids 150-275 of human ICK) against proteome databases

  • Identify proteins with significant sequence homology

  • Pay particular attention to related kinases (MAK, MOK) that share structural similarities with ICK

  • Generate a priority list of potential cross-reactants for experimental validation

• Experimental validation design:

  • Test the antibody against recombinant proteins of identified potential cross-reactants

  • Implement knockout/knockdown controls where ICK expression is eliminated

  • Compare staining patterns in cells known to express or lack ICK

  • Use peptide competition assays with both target and non-target peptides

• Multiparametric validation:

  • Correlate results from biotin-conjugated antibody with alternative detection methods

  • Compare protein detection with mRNA expression patterns

  • Implement proteomics approaches to identify all proteins captured by the antibody

• Quantitative specificity metrics:

  • Calculate signal-to-noise ratios across different sample types

  • Determine detection thresholds based on negative control samples

  • Establish calibration curves with defined specificity tolerances

  • Report the minimal detectable concentration and linear range

• Troubleshooting strategy for suspected cross-reactivity:

  • Increase stringency of washing steps (higher salt concentration, increased detergent)

  • Implement pre-absorption with suspected cross-reactants

  • Test alternative clone or epitope for comparison

  • Consider multiple antibody approach targeting different regions of ICK

This systematic approach to cross-reactivity assessment ensures that experimental results reflect true ICK expression patterns rather than artifacts from non-specific binding .

What advanced imaging analysis techniques can be applied to ICK localization studies using biotin-conjugated antibodies?

Advanced imaging analysis provides deeper insights into ICK localization and function when using biotin-conjugated antibodies:

Advanced Imaging Analysis Methodology:

• Quantitative co-localization analysis:

  • Calculate Pearson's correlation coefficient between ICK and ciliary markers

  • Implement Manders' overlap coefficient to determine fractional overlap

  • Apply intensity correlation analysis (ICA) for relationship between signal intensities

  • Establish quantitative thresholds for biological relevance of co-localization

• Super-resolution approaches:

  • Implement STORM or PALM imaging using photoswitchable fluorophores conjugated to streptavidin

  • Achieve 10-20 nm resolution of ICK localization within ciliary structures

  • Apply deconvolution algorithms to improve resolution in conventional microscopy

  • Use structured illumination microscopy (SIM) for 2x resolution improvement

• 3D reconstruction and analysis:

  • Acquire Z-stacks with step size of 0.2-0.3 μm

  • Apply 3D reconstruction algorithms with appropriate point-spread function modeling

  • Quantify volumetric distribution of ICK within cellular compartments

  • Correlate 3D distribution with functional parameters

• Temporal dynamics analysis:

  • Implement time-lapse imaging with optimized acquisition parameters

  • Apply kymograph analysis to visualize ICK movement along ciliary structures

  • Quantify trafficking rates and directional persistence

  • Correlate temporal patterns with cell cycle stages or signaling events

• Machine learning-based approaches:

  • Train neural networks to recognize specific ICK localization patterns

  • Implement automated segmentation of cellular compartments

  • Extract multi-parametric features from image datasets

  • Identify subtle phenotypic changes not apparent through visual inspection

These advanced imaging analysis techniques enable researchers to extract maximum information from ICK localization studies, providing insights into spatial relationships, temporal dynamics, and functional correlations that may not be apparent through conventional approaches .