ING2 Antibody

What is ING2 and why is it an important research target?

ING2 (Inhibitor of Growth Family, Member 2) is a member of the ING tumor suppressor family that associates with and modulates the activity of histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes. ING2 functions in critical cellular processes including DNA repair, apoptosis, and cell cycle regulation. Research has demonstrated its role in chromatin binding, autophagy, and negative regulation of intrinsic apoptotic signaling . It has gained significance as a research target due to its demonstrated function as a mediator of Transforming Growth Factor-β (TGF-β) signaling, where it promotes TGF-β-induced transcription and cell cycle arrest . These characteristics make ING2 an important subject for cancer research, epigenetic studies, and cellular signaling investigations.

How do I select the appropriate ING2 antibody for my research needs?

When selecting an ING2 antibody, consider these key technical parameters:

• Target species reactivity: Verify that the antibody has been validated for your species of interest. Available ING2 antibodies may react with human, mouse, rat, and sometimes multiple species including horse, rabbit, cow, dog, guinea pig, zebrafish, monkey, and pig .

• Clonality: Determine whether polyclonal or monoclonal antibodies better suit your experiment:

  • Polyclonal antibodies recognize multiple epitopes and may provide stronger signals

  • Monoclonal antibodies (e.g., clones 1D1, 2F5, 2G3) offer higher specificity for a single epitope

• Host species: Consider the host species (rabbit, mouse, goat) in relation to your secondary detection system and to avoid cross-reactivity in your experimental system .

• Applications: Verify validation for your specific application (Western blotting, ELISA, immunohistochemistry, immunofluorescence, immunoprecipitation) .

• Epitope location: Some antibodies target specific regions (N-terminal, C-terminal, or particular amino acid sequences like AA 1-280, AA 112-141, AA 25-74), which may be important depending on your research question .

Always review validation data and literature citations when available to ensure the antibody has demonstrated performance in applications similar to yours.

What are the optimal conditions for using ING2 antibodies in Western blotting?

For optimal Western blotting results with ING2 antibodies:

• Sample preparation:

  • Use RIPA or NP-40 buffer with protease inhibitors for cell/tissue lysis

  • Include phosphatase inhibitors if studying phosphorylation states of ING2

  • Heat samples at 95°C for 5 minutes in reducing SDS sample buffer

• Gel selection and transfer:

  • Use 10-12% SDS-PAGE gels (ING2 has a molecular weight of approximately 33 kDa)

  • Transfer to PVDF or nitrocellulose membranes at 100V for 60-90 minutes

• Antibody dilutions and incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Use ING2 antibody at 1:500-1:2000 dilution as recommended

  • Incubate overnight at 4°C for optimal results

  • Use TBS with 0.05-0.1% Tween-20 for washing steps

• Detection considerations:

  • Select appropriate HRP-conjugated secondary antibody based on host species

  • For phospho-specific detection, BSA is preferred over milk as a blocking agent

  • When studying ING2 interactions with TGF-β signaling components, consider stripping and reprobing for related proteins like Smad2 or SnoN

• Expected results:

  • Wild-type ING2 should appear at approximately 33 kDa

  • Verify antibody specificity using positive controls and ING2 knockdown samples

How can I optimize ING2 antibody performance in immunohistochemistry?

To optimize ING2 antibody performance in immunohistochemistry:

• Fixation and antigen retrieval:

  • For FFPE tissues, use citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for antigen retrieval

  • Heat-induced epitope retrieval (HIER) at 95-98°C for 15-20 minutes typically yields better results than enzymatic methods

  • Freshly fixed tissues (24-48 hours) often provide optimal antigen preservation

• Antibody concentration and incubation:

  • Use ING2 antibody at dilutions between 1:50-1:200 as recommended

  • Overnight incubation at 4°C typically provides better signal-to-noise ratio than shorter incubations

  • Include proper negative controls (isotype control or secondary antibody only)

• Signal amplification and detection:

  • For low expression levels, consider biotin-streptavidin or tyramide signal amplification

  • For co-localization studies, use fluorescent secondary antibodies with appropriate controls

• Validation strategies:

  • Compare staining pattern across multiple ING2 antibodies targeting different epitopes

  • Include positive control tissues known to express ING2

  • For critical studies, confirm specificity using ING2 knockdown tissues/cells

• Expected localization:

  • ING2 localizes primarily to the nucleus, with potential detection in nuclear substructures

  • When validating new antibodies, verify this characteristic nuclear localization pattern

How can I study ING2's role in TGF-β signaling pathways?

To investigate ING2's role in TGF-β signaling:

• Reporter gene assays:

  • Utilize TGF-β-responsive reporters such as the 3TP-luciferase reporter containing Smad binding elements (SBEs)

  • Co-transfect cells with the reporter construct and ING2 expression plasmids

  • Treat with TGF-β ligand (typically 100-200 pM) for 16-24 hours before assessing luciferase activity

  • Include appropriate controls (empty vector, TGF-β receptor inhibitors)

• Domain function analysis:

  • Generate ING2 mutants lacking specific domains:

    • N-terminal deletion (ΔN) removing leucine zipper-like motif (amino acids 1-63)

    • C-terminal deletion (ΔC) removing PHD domain (amino acids 199-281)

    • PHD domain deletion (ΔPHD) (amino acids 199-258)

  • Assess these mutants in reporter assays to determine domain-specific contributions to TGF-β signaling

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation to detect interactions between ING2 and:

    • SnoN (a transcriptional modulator)

    • Smad2 (TGF-β-regulated transcription factor)

  • Use both overexpressed tagged proteins and endogenous proteins

  • Include RNase treatment to rule out RNA-mediated interactions

• Gene expression analysis:

  • Monitor TGF-β target genes (e.g., PAI-1) by qRT-PCR or Western blot

  • Compare responses in cells with ING2 overexpression, knockdown, or mutant expression

  • Include time-course analysis (2-48 hours) to capture early and late TGF-β responses

• Cell proliferation assays:

  • Assess how ING2 affects TGF-β-mediated growth inhibition using:

    • BrdU incorporation

    • Cell counting

    • Colony formation assays

  • Determine how ING2 affects sensitivity to different TGF-β concentrations

What approaches can I use to study ING2's chromatin remodeling functions?

To investigate ING2's chromatin remodeling functions:

• Chromatin immunoprecipitation (ChIP):

  • Perform ChIP with ING2 antibodies to identify genomic binding sites

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

  • Look for co-occupancy with histone marks (H3K4me3, H3K27ac) and TGF-β-regulated transcription factors

  • Compare binding patterns before and after TGF-β stimulation

• Histone modification analysis:

  • Examine how ING2 overexpression or knockdown affects:

    • H3K4 methylation

    • Histone acetylation at TGF-β target genes

  • Use Western blotting with histone modification-specific antibodies

  • Perform ChIP for histone marks at specific promoters

• Chromatin accessibility assays:

  • Use ATAC-seq or DNase-seq to determine how ING2 affects chromatin accessibility

  • Focus on TGF-β responsive genomic regions

  • Compare wild-type ING2 versus PHD domain mutants

• Protein complex identification:

  • Perform tandem affinity purification of ING2 followed by mass spectrometry

  • Identify components of ING2-containing HAT or HDAC complexes

  • Verify interactions with co-immunoprecipitation

  • Determine how TGF-β signaling affects complex composition

• Live-cell imaging:

  • Use fluorescently tagged ING2 to track dynamics at chromatin

  • Perform fluorescence recovery after photobleaching (FRAP) to measure residence time on chromatin

  • Compare dynamics before and after TGF-β stimulation

How can I address specificity concerns when working with ING2 antibodies?

To address specificity concerns:

• Validation controls:

  • Genetic approaches: Use ING2 knockout/knockdown cells or tissues as negative controls

  • Peptide competition: Pre-incubate antibody with the immunizing peptide before application

  • Multiple antibodies: Use antibodies targeting different ING2 epitopes and compare staining patterns

  • Heterologous expression: Test antibody on cells overexpressing tagged ING2

• Cross-reactivity assessment:

  • Test for cross-reactivity with other ING family members (ING1, ING3-5)

  • For polyclonal antibodies, consider affinity purification against recombinant ING2

  • When detecting endogenous ING2, include positive control samples with known expression

• Technical approaches to improve specificity:

  • Optimize antibody concentration (use titration experiments)

  • Increase stringency of washing steps (higher salt concentration, longer washes)

  • Use more selective blocking agents (specific for your application)

  • Consider monoclonal antibodies for higher specificity in challenging applications

• Application-specific considerations:

  • For Western blotting: Verify band size (33 kDa for wild-type ING2)

  • For immunoprecipitation: Include isotype control antibodies

  • For immunohistochemistry: Include absorption controls with recombinant protein

What are common challenges when detecting interactions between ING2 and its binding partners?

Common challenges and solutions:

• Transient or weak interactions:

  • Use chemical crosslinking (formaldehyde, DSS, or BS3) before lysis

  • Try proximity ligation assay (PLA) for detecting in situ protein interactions

  • Consider bimolecular fluorescence complementation (BiFC) for live cell analysis

• Buffer composition challenges:

  • Test multiple lysis buffers (RIPA vs. NP-40 vs. digitonin-based)

  • Adjust salt concentration to optimize interaction stability

  • Include phosphatase inhibitors when studying phosphorylation-dependent interactions

  • Consider the presence/absence of detergents that might disrupt certain interactions

• Nuclear protein extraction issues:

  • Use specialized nuclear extraction protocols for efficient ING2 isolation

  • Consider DNase/RNase treatment to release chromatin-bound proteins

  • For histone-associated interactions, include histone deacetylase inhibitors

• Post-translational modifications:

  • For studying interactions with SnoN and Smad2, consider how TGF-β treatment affects:

    • Phosphorylation states

    • Protein stability

    • Nuclear-cytoplasmic distribution

  • Time course experiments may be necessary to capture dynamic interactions

• Detection sensitivity:

  • For endogenous interactions, optimize antibody combinations that don't cross-react

  • Consider using more sensitive detection methods (e.g., proximity-based assays)

  • For mass spectrometry, use SILAC or TMT labeling to distinguish specific from non-specific interactions

How should I interpret conflicting results between different ING2 antibodies?

When faced with conflicting results:

• Epitope considerations:

  • Determine the epitopes recognized by each antibody (N-terminal, C-terminal, internal)

  • Conflicting results may reflect detection of different:

    • ING2 isoforms

    • Post-translational modifications

    • Protein-protein interaction states that mask certain epitopes

• Methodological approach:

  • Compare antibody performance across multiple techniques:

    • If conflict exists only in one technique (e.g., IHC), it may reflect fixation/processing sensitivity

    • If conflict exists across techniques, consider fundamental specificity issues

  • Test antibodies on recombinant ING2 constructs with known modifications/truncations

• Validation strategies:

  • Use genetic models (CRISPR knockout, siRNA) to confirm specificity

  • Perform rescue experiments with ING2 reexpression

  • Consider advanced approaches like mass spectrometry to verify antibody targets

• Interpretation framework:

  • Create a decision tree based on:

    • Antibody validation documentation

    • Published literature using each antibody

    • Technical controls performed in your experiments

  • Weight evidence based on rigor of controls and consistency across techniques

• Reporting guidelines:

  • Document all antibodies used (catalog numbers, dilutions, lots)

  • Clearly describe conflicting results in publications

  • Consider showing results from multiple antibodies when conflicts exist

  • Discuss potential biological explanations for differences

How can I differentiate between direct and indirect effects of ING2 in TGF-β signaling experiments?

To differentiate direct from indirect effects:

• Temporal analysis:

  • Perform detailed time-course experiments:

    • Immediate early responses (0-2 hours) more likely reflect direct effects

    • Delayed responses (>4 hours) may indicate secondary effects

  • Use protein synthesis inhibitors (cycloheximide) to block secondary responses requiring new protein synthesis

• Domain mutation approaches:

  • Compare wild-type ING2 with domain mutants in rescue experiments

  • PHD domain mutants particularly important as this domain is critical for TGF-β signaling

  • Effects rescued by wild-type but not by PHD mutants likely represent direct mechanisms

• Chromatin occupancy analysis:

  • Perform ChIP-seq for ING2 and Smad proteins

  • Sites co-occupied by both factors more likely represent direct regulatory targets

  • Integrate with RNA-seq after short TGF-β treatment to identify immediate transcriptional effects

• Biochemical interaction assays:

  • Use in vitro binding assays with purified components to confirm direct interactions

  • Reconstitute minimal systems to determine sufficiency for specific effects

  • Consider in vitro transcription systems to test direct transcriptional regulation

• Genetic epistasis experiments:

  • Perform double knockdown/knockout experiments:

    • ING2 + Smad2/3

    • ING2 + SnoN

  • Compare phenotypes to determine pathway relationships

  • Use rescue experiments with mutant proteins resistant to siRNA targeting

Table 1: Experimental Approaches to Distinguish Direct vs. Indirect ING2 Effects

What statistical approaches should I use when analyzing ING2 expression or functional data?

Appropriate statistical approaches depend on the experimental design:

• Comparative expression analysis:

  • For comparing ING2 levels across conditions:

    • Use t-tests for two-group comparisons (if normally distributed)

    • Use ANOVA followed by post-hoc tests for multiple group comparisons

    • Consider non-parametric alternatives (Mann-Whitney, Kruskal-Wallis) for non-normal distributions

  • Calculate appropriate effect sizes (Cohen's d, fold change)

  • Report both raw p-values and adjusted p-values for multiple comparisons

• Correlation analysis:

  • For examining relationships between ING2 and other variables:

    • Use Pearson correlation for linear relationships (if normally distributed)

    • Use Spearman correlation for non-parametric or non-linear relationships

    • Consider partial correlations to control for confounding variables

  • Report both correlation coefficients and confidence intervals

• Functional assays:

  • For reporter gene assays:

    • Normalize to appropriate controls (renilla luciferase, β-galactosidase)

    • Use fold-change relative to unstimulated or vector control

    • Apply two-way ANOVA to assess interaction between ING2 expression and TGF-β treatment

  • For proliferation assays:

    • Consider area-under-curve (AUC) analysis for growth curves

    • Calculate EC50 values to compare sensitivities to TGF-β

• Omics data integration:

  • For ChIP-seq or RNA-seq:

    • Apply multiple testing correction (FDR, Bonferroni)

    • Use enrichment analyses for pathway/GO term identification

    • Consider integrated approaches (GSEA, network analysis)

  • Report both statistical significance and biological significance (fold change)

• Power and sample size:

  • Conduct a priori power analysis to determine appropriate sample sizes

  • Report effect sizes alongside p-values

  • Consider biological replicates (independent experiments) vs. technical replicates

How can I investigate the role of ING2 in TGF-β-mediated tumor suppression versus tumor promotion?

To investigate this dual role:

• Cell context-dependent studies:

  • Compare ING2's effects in:

    • Normal epithelial cells (e.g., Mv1Lu) where TGF-β is typically growth inhibitory

    • Advanced cancer cell lines where TGF-β may promote metastasis

    • Early versus late-stage cancer models

  • Assess canonical growth inhibition versus non-canonical pathways

• Molecular mechanism dissection:

  • Analyze ING2 interactions with specific Smad complexes:

    • Growth-inhibitory complexes (Smad3/4 with p15, p21 promoters)

    • EMT-promoting complexes (Smad3/4 with SNAI1, ZEB1 promoters)

  • Determine if ING2 differentially regulates these distinct transcriptional programs

• In vivo models:

  • Generate tissue-specific ING2 knockout or overexpression mouse models

  • Study effects on:

    • Primary tumor growth

    • Metastatic potential

    • TGF-β responsiveness in different tumor stages

  • Use genetic crosses with established TGF-β pathway mutant models

• Epigenetic landscape analysis:

  • Perform integrated epigenomic profiling:

    • ChIP-seq for ING2, Smads, and histone modifications

    • ATAC-seq for chromatin accessibility

    • DNA methylation analysis

  • Compare patterns between normal and transformed states

  • Identify switches in ING2 genomic targeting during malignant progression

• Therapeutic implications:

  • Test ING2 modulation in combination with TGF-β pathway inhibitors

  • Assess stage-specific responses to determine optimal intervention points

  • Evaluate biomarkers that predict context-dependent functions

What emerging technologies can enhance ING2 antibody-based research?

Emerging technologies with potential applications:

• Proximity-based protein interaction methods:

  • BioID or TurboID fusion proteins to identify ING2 proximity partners

  • APEX2-based proximity labeling for subcellular compartment-specific interactors

  • Split-BioID to capture condition-specific interactions (e.g., only upon TGF-β stimulation)

• Advanced microscopy approaches:

  • Super-resolution microscopy (STORM, PALM, SIM) to visualize ING2 nuclear distribution

  • Lattice light-sheet microscopy for dynamic tracking of ING2 in living cells

  • Single-molecule tracking to measure ING2-chromatin binding kinetics

• CRISPR-based technologies:

  • CUT&RUN or CUT&Tag as antibody-based alternatives to traditional ChIP

  • CRISPR activation/inhibition of ING2 for functional genomics

  • CRISPR base editing to introduce specific mutations in endogenous ING2

• Antibody engineering approaches:

  • Nanobodies against ING2 for improved penetration and reduced background

  • Intrabodies for tracking and manipulating ING2 in living cells

  • Bispecific antibodies to detect ING2 in complex with specific partners

• Mass cytometry and spatial proteomics:

  • CyTOF with ING2 antibodies for single-cell protein quantification

  • Imaging mass cytometry or CODEX for spatial context of ING2 expression

  • Hyperplexed immunofluorescence to map ING2 in relation to multiple markers

Table 2: Comparative Analysis of Emerging Technologies for ING2 Research