ING4 Antibody, FITC conjugated

What is ING4 and why is it an important research target?

ING4 functions as a tumor suppressor protein and is a component of the HBO1 complex, which specifically mediates acetylation of histone H3 at 'Lys-14' (H3K14ac) and has reduced activity toward histone H4. Through chromatin acetylation, ING4 may function in DNA replication. The protein inhibits tumor progression by modulating the transcriptional output of signaling pathways that regulate cell proliferation. ING4 can suppress brain tumor angiogenesis through transcriptional repression of RELA/NFKB3 target genes when complexed with RELA. Additionally, it may specifically suppress loss of contact inhibition elicited by activated oncogenes such as MYC and repress hypoxia inducible factor's (HIF) activity by interacting with HIF prolyl hydroxylase 2 (EGLN1). ING4 can also enhance apoptosis induced by serum starvation in mammary epithelial cell lines . Given these important functions in tumor suppression and cellular regulation, ING4 represents a significant target for cancer research and therapeutic development.

What are the key specifications of commercially available ING4-FITC antibodies?

Commercial ING4 antibodies conjugated to FITC typically present the following specifications:

This information is critical for researchers to ensure compatibility with their experimental systems and to properly interpret results .

How is FITC optimally conjugated to ING4 antibodies?

The conjugation of FITC to antibodies, including ING4 antibodies, requires specific conditions for optimal results. The process involves several key parameters:

• Purity of starting material: A relatively pure IgG fraction, obtained through DEAE Sephadex chromatography, provides the best foundation for conjugation.

• Reaction conditions: Maximal labeling is achieved under the following conditions:

  • pH: 9.5 (optimal alkaline environment)

  • Temperature: Room temperature (20-25°C)

  • Protein concentration: 25 mg/ml (initial)

  • Reaction time: 30-60 minutes

• Post-conjugation purification: Separation of optimally labeled antibodies from under- and over-labeled proteins is achieved through gradient DEAE Sephadex chromatography.

• Fluorescein/Protein (F/P) ratio: The reaction conditions should be monitored to achieve the desired F/P ratio, which is critical for downstream applications .

This methodology ensures the production of high-quality FITC-conjugated ING4 antibodies that maintain their specificity and functionality while providing the fluorescent properties required for detection.

What are the recommended storage conditions for maintaining activity of FITC-conjugated ING4 antibodies?

To maintain optimal activity of FITC-conjugated ING4 antibodies, the following storage conditions are recommended:

• Short-term storage: Aliquot and store at -20°C.

• Light sensitivity: FITC is photosensitive; therefore, protect the conjugate from excessive exposure to light during both storage and use.

• Freeze/thaw cycles: Avoid repeated freeze/thaw cycles, as these can degrade both the antibody and the fluorophore.

• Buffer conditions: Store in appropriate buffer (typically 0.01 M PBS, pH 7.4, with preservatives like 0.03% Proclin-300 and stabilizers like 50% Glycerol).

• Working dilutions: Prepare working dilutions fresh before use rather than storing diluted antibody for extended periods.

Proper storage is essential for maintaining both the immunoreactivity of the antibody and the fluorescence intensity of the FITC conjugate, which directly impacts experimental consistency and reproducibility .

What are the validated applications for ING4 antibody-FITC conjugates in cellular research?

FITC-conjugated ING4 antibodies are validated for multiple cellular research applications, each with specific methodological considerations:

• Immunofluorescence (IF): Used to visualize the subcellular localization of ING4 in fixed cells, particularly useful for studying its nuclear localization and interactions with chromatin-modifying complexes.

• Immunocytochemistry (ICC): Applied to detect ING4 in cultured cells, providing insights into expression levels and distribution patterns under various experimental conditions.

• Immunohistochemistry (IHC): Employed for detection of ING4 in tissue sections, valuable for tumor microarray analysis and comparative studies of normal versus malignant tissues.

• Flow Cytometry (FACS): Utilized for quantitative analysis of ING4 expression in cell populations, enabling correlation with other cellular parameters and sorting of specific cell populations .

For each application, optimization of antibody concentration, incubation conditions, and appropriate controls is essential for reliable results. Typically, researchers should establish optimal dilutions empirically for their specific experimental system, starting with manufacturer recommendations (often ranging from 1:50 to 1:500 depending on the application).

How can antigen-conjugated fluorescent beads be used with ING4 antibodies for cell identification?

Antigen-conjugated fluorescent beads represent an advanced methodology for identifying and isolating single antibody-expressing cells when working with ING4 antibodies:

• Bead preparation: Fluorescent beads are conjugated to ING4 protein using streptavidin/avidin-biotin bridges. This typically involves biotinylation of the ING4 protein followed by incubation with streptavidin-coated fluorescent beads.

• Verification of conjugation: Successful attachment of ING4 to the bead surface can be confirmed by staining the conjugated beads with specific antibodies against ING4, followed by detection with a secondary antibody conjugated to a different fluorophore.

• Cell selection process: The ING4-conjugated beads bind to cells expressing membrane-bound antibodies specific for ING4. These bead-bound cells can then be isolated using single-cell sorting techniques.

• Single-cell processing: The isolated cells are processed for single-cell RNA retrotranscription to identify their expressed antibodies.

This methodology enables researchers to identify and select cells expressing antibodies specific to ING4 without requiring prior ex vivo growth or secondary screening of B cells, representing a significant advancement for immunological research and antibody discovery .

How does ING4 interact with the SCFJFK complex and what are the implications for experimental design?

Research has revealed that ING4 interacts with the SCFJFK E3 ligase complex, with significant implications for experimental design and interpretation:

• Interaction mechanism: ING4 associates with the SCFJFK complex through the F-box protein JFK, which serves as a substrate receptor. Specifically, the novel conserved region (NCR; amino acids 61-120) of ING4 is responsible for its interaction with JFK.

• Complex composition: Coimmunoprecipitation experiments have demonstrated that ING4 associates with JFK as well as integral components of the SCF complex, including Cul1, Skp1, and Rbx1.

• Subcellular distribution: Protein fractionation experiments by FPLC indicate that ING4 exists in high molecular weight complexes (669-2000 kDa) that overlap with JFK, Cul1, and Skp1, supporting their in vivo association.

• Biological significance: The SCFJFK-mediated destabilization of ING4 can potentiate NF-κB signaling and promote angiogenesis and metastasis in breast cancer models .

For researchers working with ING4 antibodies, these interactions necessitate careful consideration of experimental conditions, particularly when studying ING4 stability, degradation, or functional interactions. The presence of proteasome inhibitors or the analysis of ubiquitination states may be critical for comprehensive understanding of ING4 biology.

What are the considerations for using ING4-FITC antibodies in multiplex immunofluorescence experiments?

When designing multiplex immunofluorescence experiments incorporating ING4-FITC antibodies, researchers should consider several critical factors:

• Spectral overlap: FITC has excitation/emission maxima at approximately 495/519 nm. When designing multiplex panels, select additional fluorophores with minimal spectral overlap or ensure proper compensation during analysis.

• Signal intensity balancing: ING4 may have variable expression levels across different cell types or conditions. Titrate the ING4-FITC antibody to achieve signal intensities comparable to other markers in the panel.

• Antibody cross-reactivity: Validate that the antibodies in the multiplex panel do not exhibit cross-reactivity, particularly when using multiple rabbit-derived antibodies.

• Staining sequence: For sequential staining approaches, optimize the order of antibody application. Generally, lower-abundance targets (which may include ING4 in certain cell types) should be stained earlier in the sequence.

• Fixation compatibility: Ensure that the fixation and permeabilization methods are compatible with all antibodies in the panel, as some epitopes may be sensitive to particular fixatives.

• Photobleaching considerations: FITC is relatively prone to photobleaching. When designing imaging protocols, minimize exposure to excitation light and consider using anti-fade mounting media.

These considerations will help ensure robust and reproducible results when including ING4-FITC antibodies in multiplex immunofluorescence experiments, particularly for complex tissue analysis or co-localization studies.

What are common issues when using ING4-FITC antibodies and how can they be resolved?

When working with ING4-FITC conjugated antibodies, researchers may encounter several technical challenges. Here are common issues and their solutions:

For all troubleshooting scenarios, inclusion of appropriate positive and negative controls is essential for proper interpretation of results and optimization of protocols .

How can researchers optimize the Fluorescein/Protein (F/P) ratio for ING4-FITC antibodies?

The Fluorescein/Protein (F/P) ratio is a critical parameter that affects the performance of FITC-conjugated antibodies. Optimal F/P ratios for ING4-FITC antibodies typically range from 3-8 fluorescein molecules per antibody molecule. Here's how researchers can optimize this ratio:

• Reaction parameter adjustment:

  • pH: Maintain at 9.5 for optimal FITC reactivity with primary amines

  • FITC concentration: Titrate between 10-50 μg FITC per mg of antibody

  • Reaction time: Control between 30-60 minutes at room temperature

  • Protein concentration: Use 10-25 mg/ml ING4 antibody concentration

• Monitoring conjugation efficiency:

  • Spectrophotometric analysis: Calculate F/P ratio using absorbance measurements at 280 nm (protein) and 495 nm (FITC)

  • F/P Ratio calculation:
    F/P = (A495 × dilution factor) / (195 × protein concentration in mg/ml)

• Purification of optimally labeled fraction:

  • Gradient DEAE Sephadex chromatography separates antibody populations with different F/P ratios

  • Size exclusion chromatography removes free FITC

  • Antibodies with optimal F/P ratios typically elute in specific fractions

• Validation of conjugate performance:

  • Flow cytometry titration to determine optimal working concentration

  • Comparison of signal-to-noise ratio across different F/P preparations

  • Assessment of non-specific binding

Optimizing the F/P ratio is essential for balancing signal intensity against potential issues like self-quenching, increased non-specific binding, or altered antibody specificity that can occur with excessively high F/P ratios .

How can researchers accurately quantify ING4 expression using FITC-conjugated antibodies?

Accurate quantification of ING4 expression using FITC-conjugated antibodies requires carefully controlled methodologies:

• Flow cytometry quantification:

  • Use calibration beads with known quantities of FITC molecules to establish a standard curve

  • Apply compensation when using multiple fluorophores to correct for spectral overlap

  • Consider using Molecules of Equivalent Soluble Fluorochrome (MESF) values for standardization

  • Always include isotype controls conjugated to FITC to determine background fluorescence levels

• Fluorescence microscopy quantification:

  • Implement consistent image acquisition parameters (exposure time, gain, offset)

  • Use reference standards in each imaging session

  • Apply appropriate background subtraction methods

  • Consider automated analysis software for unbiased quantification of signal intensity

• Western blot with fluorescent detection:

  • Include a standard curve of recombinant ING4 protein

  • Use total protein normalization rather than single housekeeping proteins

  • Ensure linear range of detection for accurate quantification

  • Consider using near-infrared fluorescent detection systems for improved quantitative range

• Controls for quantification accuracy:

  • Include known positive and negative cell lines or tissues

  • Validate results with orthogonal methods (e.g., mRNA quantification)

  • Use ING4 knockdown or knockout samples as negative controls

  • Apply statistical analysis appropriate for the distribution of the data

These approaches enable reliable quantification of ING4 expression across different experimental systems and conditions .

What are the cutting-edge applications of ING4-FITC antibodies in cancer research?

ING4-FITC antibodies are being utilized in several innovative applications in cancer research:

• Tumor microenvironment analysis:

  • Multi-parameter flow cytometry to correlate ING4 expression with immune cell infiltration

  • Spatial analysis of ING4 expression relative to hypoxic regions in tumors

  • Investigation of ING4 regulation in response to therapeutic interventions

• Mechanistic studies of tumor suppression:

  • High-content imaging to track ING4 subcellular localization during cell cycle progression

  • Co-localization studies with HBO1 complex components to understand chromatin regulation

  • Analysis of ING4 dynamics in response to oncogenic stress

• Biomarker development:

  • Correlation of ING4 expression patterns with clinical outcomes

  • Development of ING4-based prognostic indicators

  • Identification of patient subgroups that might benefit from therapies targeting ING4-related pathways

• Angiogenesis research:

  • Investigation of ING4's role in suppressing tumor angiogenesis

  • Analysis of ING4 interaction with HIF signaling in endothelial cells

  • Examination of NF-κB pathway regulation by ING4 in tumor-associated blood vessels

• Therapeutic development:

  • Screening for compounds that stabilize ING4 against degradation by SCFJFK

  • Evaluation of ING4 status as a predictor of response to epigenetic therapies

  • Development of strategies to restore ING4 function in tumors

These applications reflect the growing importance of ING4 in understanding cancer biology and developing new therapeutic approaches, with FITC-conjugated antibodies serving as valuable tools for visualization and quantification in these research contexts .