ING3 Antibody

What is ING3 and why is it important in cancer research?

ING3 is a nuclear tumor-suppressor protein that plays a significant role in regulating cellular responses to stress, particularly in UV-induced damage. It functions as a component of the NuA4 histone acetyltransferase (HAT) complex involved in transcriptional activation of select genes through acetylation of nucleosomal histones H4 and H2A . ING3 activates apoptosis through a FAS-mediated pathway, which is vital for eliminating damaged cells and preventing cancer development .

Recent research has demonstrated that ING3 is most highly expressed in tissues with rapid cellular turnover, including small intestine, bone marrow, and epidermis . This expression pattern and its involvement in p53-mediated transcription suggest that ING3 plays critical roles in growth regulation, self-renewal, and cancer development .

Most ING3 antibodies should be stored at -20°C prior to opening. For extended storage, it's recommended to aliquot contents and freeze at -20°C or below to avoid repeated freeze-thaw cycles . Once thawed, many antibodies remain stable for several weeks at 4°C as undiluted liquids.

For example, the ING3 Antibody (NBP1-78100) is supplied in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, 0.02% sodium azide, and 50% glycerol . These components help maintain antibody stability during storage. Always centrifuge the product if not completely clear after standing at room temperature and dilute only prior to immediate use .

How do I validate the specificity of an ING3 antibody before experimental use?

Validating antibody specificity is crucial for reliable results. Here's a recommended approach:

• Western blot validation: Using positive controls (e.g., cells known to express ING3) and negative controls (e.g., cells with ING3 knockdown). A specific band should appear at approximately 47 kDa (ING3's molecular weight) .

• Overexpression control: Compare cells transfected with ING3 expression vectors to non-transfected cells. For example, one study showed detection of a band at ~55 kDa in RKO cells transfected with ING3, while control RKO cells didn't show this specific band .

• Multiple antibody approach: Use different antibodies targeting different epitopes of ING3 and compare the results.

• Immunoprecipitation followed by mass spectrometry: This can confirm that the antibody is specifically pulling down ING3 and identify any cross-reacting proteins.

• Knockdown/knockout validation: siRNA or CRISPR approaches can be used to reduce or eliminate ING3 expression, with subsequent testing showing reduced or absent signal in Western blot or immunohistochemistry .

What are the optimal conditions for using ING3 antibodies in Western blotting?

Based on the literature and product information, the following conditions have been successful for ING3 Western blot detection:

• Sample preparation:

  • Use approximately 10-20 μg of whole cell lysate per lane

  • Separate proteins on a 4-20% Tris-Glycine gel by SDS-PAGE

• Blocking:

  • 5% non-fat dry milk in TBST for 1 hour at room temperature

• Primary antibody incubation:

  • Dilution: 1:1,000 (may vary by specific antibody)

  • Incubation: Overnight at 4°C

• Secondary antibody:

  • For goat primary: Rabbit-anti-Goat IgG (H&L) (1:20,000)

  • For rabbit primary: Anti-rabbit HRP-conjugated (typically 1:5,000)

  • For mouse primary: Anti-mouse HRP-conjugated (typically 1:5,000)

• Detection:

  • Either chemiluminescence or fluorescence-based detection systems

  • IRDye™800 fluorescence imaging has been successfully used with Odyssey® Infrared Imaging System

How can I optimize ING3 immunofluorescence staining to visualize its subcellular localization?

ING3 is primarily a nuclear protein involved in chromatin remodeling, so proper immunofluorescence optimization is critical:

• Fixation:

  • 4% paraformaldehyde (10-15 minutes) followed by permeabilization with 0.1-0.5% Triton X-100

  • Alternative: Cold methanol (-20°C) for 10 minutes (may better preserve nuclear architecture)

• Blocking:

  • 5% normal serum (matching secondary antibody host) for 1 hour

• Antibody concentration:

  • Use higher dilutions than Western blot (typically 1:200 to 1:500)

  • Incubate overnight at 4°C or 2 hours at room temperature

• Nuclear counterstain:

  • DAPI is commonly used to visualize nuclei (as demonstrated in A549 cells)

• Controls:

  • Include secondary-only controls to evaluate background

  • Use cells with known high and low ING3 expression

• Confocal microscopy:

  • Recommended for better resolution of nuclear structures

Remember that ING3 is a component of the NuA4 histone acetyltransferase complex and may show punctate nuclear staining patterns corresponding to sites of active transcription or DNA repair .

How does ING3 expression differ between normal and cancerous tissues, and what are the methodological considerations for such comparisons?

Studies have revealed important differences in ING3 expression patterns:

• Tissue-specific expression:

  • ING3 is highly expressed in tissues with rapid cell turnover: small intestine, bone marrow, and epidermis

  • Expression is significantly higher in proliferating versus quiescent epithelial cells

• Cancer alterations:

  • Evidence suggests ING3 may play dual roles depending on cancer type

  • In prostate cancer, ING3 has been identified as an oncoprotein rather than a tumor suppressor

• Methodological considerations:

  • Use tissue microarrays (TMAs) for comparing multiple samples under identical conditions

  • Quantify expression using digital pathology and image analysis software

  • Include multiple normal tissue controls from various sites

  • Consider cell-type specific expression differences within heterogeneous tissues

  • Always validate IHC findings with orthogonal methods (qPCR, Western blot)

  • Account for potential isoform-specific expression differences

What methods can be used to investigate the role of ING3 in the NuA4 histone acetyltransferase complex?

The NuA4 HAT complex is critical for transcriptional regulation through histone modification. To study ING3's role:

• Co-immunoprecipitation (Co-IP):

  • Use ING3 antibodies to pull down associated proteins, then probe for other NuA4 components

  • Alternatively, IP other NuA4 components and blot for ING3

  • Cross-linking before IP can help capture transient interactions

• Chromatin Immunoprecipitation (ChIP):

  • Use ING3 antibodies to identify genomic regions where ING3 is bound

  • Sequential ChIP (re-ChIP) can determine co-occupancy with other NuA4 components

  • Combine with sequencing (ChIP-seq) for genome-wide binding profiles

• Proximity ligation assay (PLA):

  • Visualize and quantify interactions between ING3 and other NuA4 components in situ

• Histone acetyltransferase assays:

  • Compare HAT activity in the presence or absence (knockdown) of ING3

  • Measure acetylation of specific histone residues (H4, H2A) by Western blot

• Gene expression analysis after ING3 modulation:

  • RNA-seq or microarray analysis following ING3 knockdown/overexpression

  • Focus on genes known to be regulated by NuA4 complex

Remember that ING3 is specifically involved in the removal of histone H2A.Z/H2AZ1 from nucleosomes as part of a SWR1-like complex .

How can computational approaches enhance ING3 antibody research and epitope mapping?

Computational methods can significantly advance antibody-based research:

• Antibody modeling and structure prediction:

  • Homology modeling using tools like PIGS server or AbPredict algorithm can generate 3D structures of antibody variable fragments

  • Molecular dynamics simulations can refine these models

• Epitope mapping:

  • Computational prediction of linear and conformational epitopes

  • In silico alanine scanning to identify critical binding residues

  • Molecular docking to predict antibody-antigen interactions

• Specificity analysis:

  • Computational screening against human proteome to identify potential cross-reactive proteins

  • Analysis of sequence conservation across species to predict cross-reactivity

• High-throughput screening approaches:

  • Computational design of peptide arrays for epitope mapping

  • Virtual screening of antibody variants with improved specificity

A combined approach using experimental techniques (site-directed mutagenesis, STD-NMR) with computational modeling can help define the antibody-antigen interface with high precision .

What are common issues with ING3 antibody experiments and how can they be resolved?

When troubleshooting, remember that ING3 has multiple isoforms, and antibodies may have different specificities. For example, the NBP1-78100 antibody shows homology only to isoform 1 for ING3 and not isoforms 2 and 3 .

How should researchers interpret changes in ING3 localization or expression across experimental conditions?

Interpreting ING3 changes requires careful consideration:

• Subcellular localization changes:

  • Nuclear to cytoplasmic shifts may indicate disrupted function

  • Changes in nuclear punctate patterns may reflect altered chromatin association

  • Co-localization with DNA damage markers suggests recruitment to damage sites

• Expression level changes:

  • Correlate with proliferation markers (Ki-67, PCNA) as ING3 is higher in proliferating cells

  • Consider cell cycle phase as expression may vary throughout the cycle

  • Use quantitative methods (densitometry for WB, fluorescence intensity for IF)

• Functional consequences:

  • Changes in histone acetylation (especially H4 and H2A)

  • Altered expression of NuA4-regulated genes

  • Effects on apoptosis (caspase activation, BID cleavage)

  • Impact on DNA repair capacity

• Context dependency:

  • ING3 may have different roles depending on cell type and physiological context

  • Its function as tumor suppressor or oncoprotein may be tissue-dependent

What are emerging applications of ING3 antibodies in cancer research and potential therapeutic approaches?

Emerging research directions include:

• Biomarker development:

  • ING3 levels may serve as a surrogate for growth rate in tumors

  • Expression patterns could potentially differentiate cancer subtypes

  • Ratio of nuclear to cytoplasmic ING3 may have prognostic value

• Therapeutic targeting:

  • Antibody-drug conjugates targeting surface-exposed ING3 in cancer cells

  • Small molecules disrupting ING3 interactions with the NuA4 complex

  • Synthetic lethality approaches targeting cells with aberrant ING3 expression

• Combination approaches:

  • Combining ING3 targeting with DNA damage-inducing therapies

  • Exploiting synthetic lethal interactions with other chromatin modifiers

• Personalized medicine applications:

  • Stratifying patients based on ING3 expression patterns

  • Developing companion diagnostics using ING3 antibodies

How can single-cell analysis approaches enhance our understanding of ING3 function in heterogeneous tissues?

Single-cell technologies offer significant advantages:

• Single-cell RNA sequencing (scRNA-seq):

  • Reveals cell-type specific expression patterns of ING3

  • Identifies co-expression networks in different cell populations

  • Captures rare cell types with unique ING3 expression patterns

• Single-cell proteomics:

  • Mass cytometry (CyTOF) with ING3 antibodies can quantify protein at single-cell level

  • Multiplex immunofluorescence reveals co-expression with other proteins

• Spatial transcriptomics/proteomics:

  • Maps ING3 expression in tissue context while maintaining spatial information

  • Correlates ING3 levels with microenvironmental factors

• Single-cell ChIP-seq:

  • Identifies cell-specific binding patterns of ING3 to chromatin

  • Reveals heterogeneity in ING3 function across cell populations

These approaches are particularly relevant given ING3's differential expression in proliferating versus quiescent cells, which may not be captured in bulk tissue analyses .