IKBIP Antibody，FITC conjugated

What is IKBIP and what is its significance in research?

IKBIP (I kappa B kinase interacting protein), also known as IKIP, is a protein encoded by a gene located on human chromosome 12q23.1. It consists of four exons (E1, E2, E3, and E3a) which can be alternatively spliced to produce three different transcripts: IKBIP-1, IKBIP-2, and IKBIP-3 . IKBIP was first discovered and studied in 2004 by Hofer-Warbinek et al. and has gained interest as a research target due to its:

• Role in inhibiting NF-κB activation through inhibition of IKKα/β phosphorylation

• Function as a p53 target gene with proapoptotic properties

• Expression in vascular endothelial cells, with additional detection of isoform 4 in lung, kidney, spleen, thymus, and skeletal muscle

• Correlation with various cancer types and immune infiltration

Recent studies have demonstrated IKBIP's potential as a biomarker in multiple cancer types, making it an increasingly important target for antibody-based detection methods .

What are the properties of FITC-conjugated antibodies?

FITC (Fluorescein Isothiocyanate) is one of the most widely used fluorophores for antibody conjugation with the following characteristics:

What is the reactivity spectrum of commercially available IKBIP-FITC antibodies?

Based on the search results, IKBIP antibodies including FITC-conjugated versions typically demonstrate the following reactivity profile:

For the specific IKBIP Rabbit Polyclonal Antibody (FITC) found in the search results, the antibody demonstrates reactivity to human IKBIP, targeting the C-terminal epitope . It is important to verify the specific reactivity of any antibody with the manufacturer before use in critical experiments.

How is IKBIP expression linked to cancer progression and prognosis?

Recent comprehensive pan-cancer analyses have revealed important correlations between IKBIP expression and cancer:

IKBIP expression is significantly elevated in multiple cancer types compared to normal tissues, suggesting a potential role as an oncogene . A 2023 study demonstrated that:

• IKBIP expression is negatively associated with prognosis in several major cancer types

• IKBIP expression was linked to tumor mutational burden (TMB) in 13 cancer types and microsatellite instability (MSI) in seven cancer types

• Various cancer types exhibit unique tumor-infiltrating immune cell profiles associated with IKBIP expression

In esophageal squamous cell carcinoma (ESCC), high IKBIP expression has been shown to:

• Promote tumor cell proliferation and migration

• Inhibit apoptosis

• Induce G1/S phase arrest

• Activate the AKT signaling pathway

These findings collectively suggest that IKBIP may serve as both a prognostic biomarker and potential therapeutic target across multiple cancer types .

How does IKBIP affect immune cell infiltration in the tumor microenvironment?

Analysis of the tumor immune microenvironment has revealed significant correlations between IKBIP expression and immune cell infiltration:

• Strong positive correlation between IKBIP expression and infiltrating immune cells in multiple cancer types, including:

  • B cells in 12 cancer types

  • CD4+ T cells in 13 cancer types

  • CD8+ T cells in 23 cancer types

  • Macrophages in 23 cancer types

  • Neutrophils in 24 cancer types

  • Dendritic cells in 24 cancer types

• IKBIP expression was negatively correlated with immune cell subtypes in COAD, LGG, BLCA, PRAD, STAD, BRCA, and READ, while showing positive correlation in THYM, OV, and LAML tissues

• The strongest correlations were observed with Th2 cells and CLP cells across various malignancies

This immunomodulatory role of IKBIP suggests it may participate in shaping the tumor immune microenvironment, potentially creating an immunosuppressive condition that facilitates tumor growth and progression .

What signaling pathways does IKBIP regulate in cancer development?

Research findings indicate that IKBIP influences several key signaling pathways involved in cancer progression:

• AKT Signaling Pathway:

  • IKBIP overexpression significantly increases phosphorylated AKT (p-AKT) expression

  • This activation promotes cell proliferation and migration in cancer cells

  • When the AKT pathway is inhibited using LY294002, the tumor-promoting effects of IKBIP are significantly reduced

• Cell Cycle Regulation:

  • IKBIP overexpression increases expression of cell cycle-related proteins:

    • C-myc

    • Cyclin D1

    • CDK2

    • CDK4

  • This leads to enhanced cell proliferation and disrupted cell cycle control

• EMT and Migration Pathways:

  • IKBIP influences expression of:

    • E-cadherin (decreased)

    • Vimentin (increased)

    • MMP2 (increased)

  • These changes promote epithelial-to-mesenchymal transition and enhanced migration capabilities

• Immune-Related Pathways:

  • IKBIP is associated with modulation of:

    • ECM-receptor interaction

    • NOD-like receptor signaling

    • Chemokine signaling pathway

    • Cytokine receptor interaction

These findings collectively demonstrate IKBIP's multifaceted role in promoting cancer development through various molecular mechanisms .

What is the optimal protocol for FITC conjugation to IKBIP antibodies?

The following protocol describes the standard method for conjugating FITC to antibodies, which can be applied to IKBIP antibodies:

Materials Required:

• Purified IKBIP antibody (1-2 mg/ml)

• FITC labeling buffer (pH 9.2)

• 5 mg/ml FITC (isomer I) in anhydrous DMSO

• Final dialysis buffer

• Dialysis tubing/cassettes

Protocol Steps:

• Dialyze purified antibody against 500 ml FITC labeling buffer at 4°C with 2-3 changes over 2 days (≥4 hr between buffer changes)

• Determine antibody concentration based on A280

• Add 20 μl of 5 mg/ml FITC in DMSO for each milligram of antibody

• Incubate for 2 hours at room temperature

• Remove unbound FITC by dialysis against 500 ml final dialysis buffer at 4°C with 2-3 changes over 2 days

• Determine the FITC/antibody ratio as follows:

  • Dilute FITC-IgG complex with dialysis buffer so that A280 < 2.0

  • Measure A280 and A492

  • Calculate protein concentration: Protein (mg/ml) = [A280 - (0.35 × A492)] × dilution factor × 1.4

  • Calculate moles of FITC: Moles FITC = (A492 × dilution factor)/(0.69 × 10^5)

  • Determine F/P ratio: F/P ratio = (Moles FITC)/(Protein concentration/1.5 × 10^5)

An optimal F/P ratio of 5-6:1 is recommended for most flow cytometry applications .

What are the recommended dilutions and applications for IKBIP-FITC antibodies?

Based on the search results, the following recommendations can be made for IKBIP-FITC antibody applications:

For immunofluorescence applications, the following protocol is recommended:

• Fix cells with appropriate fixative (commonly methanol or 4% paraformaldehyde)

• Add 2 ml blocking solution (PBS containing 10% FBS) and incubate for 20 minutes at room temperature

• Remove blocking solution and add 1 ml of PBS/10% FBS containing IKBIP-FITC antibody (1:500 dilution)

• Incubate for 1 hour at room temperature in the dark

• Wash cells 2 × 5 minutes with PBS

• Observe cells with a fluorescence microscope equipped with a FITC filter

Important: Do not freeze IKBIP-FITC antibodies and protect from continuous exposure to light to prevent loss of fluorescence .

How can I prevent photobleaching when using FITC-conjugated IKBIP antibodies?

FITC is particularly susceptible to photobleaching, which can significantly impact experimental results. The following measures can help minimize this issue:

• Storage considerations:

  • Store FITC-conjugated antibodies at 4°C in the dark

  • Avoid repeated freeze-thaw cycles

  • Add antifade agents to storage buffer

  • Consider aliquoting to minimize exposure during usage

• During experiments:

  • Minimize exposure to excitation light

  • Use antifade mounting media for microscopy samples

  • Adjust illumination intensity to the minimum required for visualization

  • Use neutral density filters to reduce excitation intensity

  • Consider acquiring data quickly or using time-lapse with intervals to reduce continuous exposure

• Alternative approaches:

  • For long-duration imaging experiments or microscopic analyses with high exposure times, consider Cyanine 5.5 labeled secondary antibodies, which offer excellent photostability and greater resistance to photobleaching compared with FITC

  • Use image acquisition software with photobleaching correction capabilities

These precautions will help maintain signal integrity throughout your experiments with FITC-conjugated IKBIP antibodies.

What are common issues with IKBIP-FITC antibody staining and how can they be resolved?

The following table outlines common problems encountered when using FITC-conjugated antibodies, including those targeting IKBIP, along with potential solutions:

When troubleshooting, always include appropriate positive and negative controls. For IKBIP-FITC antibodies, positive controls could include tissues known to express IKBIP such as vascular endothelial cells or specific cancer tissues with confirmed IKBIP expression .

What controls should be included when validating IKBIP-FITC antibody specificity?

To ensure experimental rigor when working with IKBIP-FITC antibodies, the following controls should be included:

• Positive tissue controls:

  • Human or mouse skeletal muscle, spleen, thymus tissues (known to express IKBIP)

  • Cancer tissues with confirmed IKBIP overexpression (e.g., ESCC or glioblastoma)

• Negative controls:

  • Isotype control antibody with FITC conjugation (same host species as the IKBIP antibody)

  • Secondary antibody-only control (if using indirect detection)

  • Unstained control samples

• Validation controls:

  • Competitive inhibition with recombinant IKBIP protein

  • siRNA or shRNA knockdown of IKBIP (significantly reduced signal should be observed)

  • IKBIP overexpression (increased signal should be observed)

  • Western blot confirmation of expected molecular weight (~40 kDa)

• Technical controls for FITC conjugation:

  • Measurement of fluorochrome/protein (F/P) ratio (optimal: 5-6:1)

  • Spectrophotometric analysis to confirm successful conjugation

How do I optimize IKBIP-FITC antibody concentration for different applications?

Optimizing antibody concentration is critical for achieving specific staining with minimal background. For IKBIP-FITC antibodies, the following titration approach is recommended:

General Titration Protocol:

• Prepare a series of antibody dilutions (e.g., 1:50, 1:100, 1:200, 1:500, 1:1000)

• Apply each dilution to identical samples known to express IKBIP

• Process all samples identically (same incubation times, washing steps, etc.)

• Evaluate staining intensity and background for each dilution

• Select the highest dilution that maintains specific signal with minimal background

Application-Specific Recommendations:

Important considerations:

• For some applications, a suboptimal F/P ratio may require adjustment of antibody concentration

• Sample preparation methods may affect antibody penetration and binding efficiency

• The optimal concentration may vary between different tissue types or cell lines

• Document all optimization experiments for reproducibility

Through systematic titration experiments, researchers can identify the optimal IKBIP-FITC antibody concentration that provides maximum specific signal with minimal background across different applications.

How is FITC-conjugated IKBIP antibody used in cancer research?

FITC-conjugated IKBIP antibodies are becoming increasingly valuable tools in cancer research, particularly in the following applications:

• Tumor Classification and Characterization:

  • Detection of IKBIP expression across different cancer types

  • Correlation of expression levels with tumor grade and stage

  • Identification of cancer subtypes based on IKBIP expression patterns

• Prognostic Biomarker Studies:

  • Flow cytometric analysis of IKBIP expression in patient samples

  • Correlation with clinical outcomes and survival data

  • Stratification of patients into risk categories based on IKBIP expression levels

• Immunological Research:

  • Analysis of IKBIP's relationship with immune cell infiltration

  • Investigation of IKBIP's role in modulating the tumor immune microenvironment

  • Multiplexed immunofluorescence with other immune markers to assess spatial relationships

• Therapeutic Target Validation:

  • Monitoring IKBIP expression changes in response to experimental therapies

  • Screening for compounds that modulate IKBIP expression or function

  • Assessment of drug sensitivity based on IKBIP expression levels

Recent findings have demonstrated that IKBIP expression correlates with drug sensitivity to several compounds including simvastatin, P-529, sulforaphane, teratinib, and midostatin, while showing negative correlation with DOLASTATIN 10, BMS-387032, Tamoxifen, and several other drugs .

What methodological improvements have enhanced FITC-conjugated antibody performance?

Recent advancements have improved the performance and utility of FITC-conjugated antibodies, including those targeting IKBIP:

• Optimized Conjugation Chemistry:

  • Improved control of F/P ratio for enhanced detection sensitivity

  • Development of site-specific conjugation methods to maintain antibody function

  • Standardized protocols for achieving optimal labeling efficiency

• Enhanced Stability Solutions:

  • Addition of stabilizing agents to reduce photobleaching

  • Improved buffer formulations for long-term storage

  • Development of photostabilizers compatible with live-cell imaging

• Advanced Detection Systems:

  • Higher sensitivity detectors in flow cytometry and imaging systems

  • Improved filter sets to maximize FITC signal detection while minimizing autofluorescence

  • Computational methods for signal enhancement and background reduction

• Multiplexing Capabilities:

  • Compatibility with other fluorophores for simultaneous detection of multiple targets

  • Carefully selected fluorophores to minimize spectral overlap, such as TRITC, Cyanine 3, Texas Red, and Cyanine 5

  • Advanced unmixing algorithms for improved separation of overlapping signals

These methodological improvements have significantly enhanced the utility of FITC-conjugated antibodies in both basic research and clinical applications, enabling more sensitive and specific detection of targets like IKBIP.

How does IKBIP expression correlate with drug sensitivity in cancer research?

Recent studies have investigated the relationship between IKBIP expression and drug sensitivity, revealing potential therapeutic implications:

• Positive Correlations:
  IKBIP expression showed positive correlation with sensitivity to:

  • Simvastatin (strongest positive correlation)

  • P-529

  • Sulforaphane

  • Teratinib

  • Midostatin

• Negative Correlations:
  IKBIP expression showed negative correlation with sensitivity to:

  • DOLASTATIN 10 (strongest negative correlation)

  • BMS-387032

  • Tamoxifen

  • EMD-534085

  • Vinorelbine

  • TYROTHRICIN

  • Barasertib

  • Homoharringtonine

  • ARQ-621

  • Paclitaxel

  • SR16157

  • PF-2771

  • ARRY-520 Isomer A

• Pathway-Specific Targeting:

  • AKT pathway inhibitors (e.g., LY294002) could effectively counteract the tumor-promoting effects of IKBIP overexpression

  • The efficacy of PI3K/AKT signaling inhibitors may be dependent on IKBIP expression levels

These findings suggest that IKBIP expression status could potentially serve as a predictive biomarker for drug response, guiding personalized treatment strategies in cancer therapy. Further research using FITC-conjugated IKBIP antibodies in drug screening and patient stratification could advance this promising area of investigation.

What emerging applications are being developed for IKBIP-FITC antibodies?

Several innovative applications for IKBIP-FITC antibodies are emerging in the research landscape:

• Single-Cell Analysis:

  • Integration with single-cell RNA sequencing to correlate protein expression with transcriptomic profiles

  • Application in mass cytometry (CyTOF) for high-dimensional analysis of IKBIP in cellular subpopulations

  • Spatial transcriptomics combined with IKBIP immunofluorescence to understand tissue-specific expression patterns

• Liquid Biopsy Development:

  • Detection of IKBIP in circulating tumor cells using flow cytometry

  • Analysis of IKBIP expression in exosomes as potential cancer biomarkers

  • Correlation of IKBIP levels in body fluids with tumor burden and treatment response

• Therapeutic Monitoring:

  • Real-time assessment of IKBIP modulation during experimental therapies

  • Development of companion diagnostics for potential IKBIP-targeting treatments

  • Monitoring immune infiltration changes in response to immunotherapy using multiplexed panels including IKBIP

• Advanced Imaging Applications:

  • Implementation in super-resolution microscopy for subcellular localization studies

  • Intravital imaging to track IKBIP expression in animal models

  • Correlative light and electron microscopy to link IKBIP expression with ultrastructural features

These emerging applications highlight the continuing importance of IKBIP-FITC antibodies in advancing our understanding of IKBIP's role in health and disease.

What are the latest research findings on IKBIP as a therapeutic target?

Recent studies have revealed promising insights into IKBIP's potential as a therapeutic target:

• Cancer Therapy Implications:

  • IKBIP promotes tumor development via the AKT signaling pathway in esophageal squamous cell carcinoma, suggesting AKT inhibitors may be effective in IKBIP-overexpressing tumors

  • IKBIP expression correlates with immunosuppressive environments, indicating potential synergy with immunotherapy approaches

  • The correlation between IKBIP and drug sensitivity suggests possibilities for combination therapies

• Pathway-Specific Interventions:

  • Inhibition of the AKT pathway using LY294002 significantly reduced the tumor-promoting effects of IKBIP overexpression

  • IKBIP's role in inhibiting NF-κB activation provides another potential therapeutic avenue

  • IKBIP's proapoptotic function in certain contexts suggests context-dependent targeting strategies

• Biomarker Development:

  • IKBIP expression has been linked to tumor mutational burden (TMB) in 13 cancers and microsatellite instability (MSI) in seven cancers, which may inform immunotherapy selection

  • Strong correlations with immune cell infiltration patterns suggest IKBIP as a potential predictor of immunotherapy response

These findings collectively suggest that IKBIP represents a promising therapeutic target and biomarker for cancer treatment, warranting further investigation using tools such as FITC-conjugated IKBIP antibodies for detection and monitoring.