ILK Antibody

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

Integrin-linked kinase (ILK) is a multifunctional molecular actor in cell–matrix interactions, cell adhesion, and anchorage-dependent cell growth. It combines functions of a signal transductor and a scaffold protein through its interaction with integrins, facilitating protein recruitment within the ILK–PINCH–Parvin complex (IPP). ILK is involved in crucial cellular processes including proliferation, survival, differentiation, migration, invasion, and angiogenesis . The importance of ILK is underscored by genetic analyses in various organisms showing that depletion or dysregulation leads to severe defects in integrin-containing cytoskeleton structure and cell adhesion dynamics . ILK's role in pathological conditions, including cancer progression and cardiovascular disorders, makes it a significant target for therapeutic intervention studies.

What are the major applications of ILK antibodies in research?

ILK antibodies are versatile tools applied across multiple experimental techniques:

The application versatility allows researchers to investigate ILK expression, localization, interactions, and functions across different experimental systems .

How do I choose between monoclonal and polyclonal ILK antibodies?

The choice depends on your specific research needs:

Monoclonal ILK antibodies:

• Offer higher specificity against a single epitope (e.g., clone 443208 or 1B22)

• Provide consistent lot-to-lot reproducibility

• Ideal for applications requiring high specificity, such as distinguishing between closely related proteins

• Better for quantitative analyses where consistent recognition is critical

Polyclonal ILK antibodies:

• Recognize multiple epitopes on the ILK protein

• Often provide stronger signals due to multiple binding sites

• Better for applications like immunoprecipitation and detection of denatured proteins

• May offer broader reactivity across species due to recognition of conserved epitopes

For critical experiments, validation with both types may provide complementary information and confirm results .

How should I validate the specificity of an ILK antibody?

A comprehensive validation approach should include:

• Positive and negative controls:

  • Positive: Cell lines known to express ILK (HeLa, MCF-7, C2C12, NIH3T3)

  • Negative: ILK knockout/knockdown cells or tissues

• Western blot analysis:

  • Confirm the expected molecular weight (51-59 kDa)

  • Use gradient loading to assess sensitivity (5-50 μg total protein)

• Peptide competition assay:

  • Pre-incubate antibody with specific ILK peptide immunogen

  • Signal should be significantly reduced or eliminated

• Cross-reactivity assessment:

  • Test reactivity against ILK from different species if working across species

• Isotype control experiments:

  • Use matching isotype control antibodies at the same concentration to identify non-specific binding

As demonstrated in knockout studies, proper ILK antibody validation should show complete absence of signal in ILK-deficient samples, confirming antibody specificity .

How can I optimize Western blot conditions for ILK detection?

For optimal ILK detection in Western blot experiments:

• Sample preparation:

  • Use RIPA or specialized lysis buffers with protease/phosphatase inhibitors

  • For phospho-ILK detection, add phosphatase inhibitors (sodium pyrophosphate, β-glycerophosphate, Na₃VO₄)

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels

  • Load 15-50 μg of total protein for cell lysates

• Transfer conditions:

  • PVDF membrane is recommended for ILK detection

  • For phospho-ILK detection, wet transfer is preferable

• Blocking and antibody incubation:

  • Optimize blocking with 5% non-fat milk or BSA (BSA preferred for phospho-specific antibodies)

  • Incubate with primary antibody at recommended dilution (typically 0.04-1 μg/ml or 1:500-1:2000)

  • Incubate with appropriate HRP-conjugated secondary antibody

• Detection:

  • Use ECL for standard detection

  • For low expression samples, consider enhanced chemiluminescence substrates

For reproducible results, follow these optimization steps and include positive controls like HeLa, MCF-7, or C2C12 cell lysates that reliably express ILK .

What considerations are important for ILK immunocytochemistry/immunofluorescence experiments?

For successful immunocytochemistry/immunofluorescence detection of ILK:

• Fixation method:

  • Optimize between paraformaldehyde (4%, 15-30 min) for structure preservation or acetone for better epitope accessibility

  • Different fixatives may affect antibody recognition of ILK

• Permeabilization:

  • 0.1-0.5% Triton X-100 for cytoplasmic and nuclear ILK

  • Milder detergents (0.1% saponin) for membrane-associated ILK

• Antibody concentration:

  • Typically 1-2 μg/ml for primary ILK antibodies (1:100-1:200 dilution)

  • Include isotype controls at equivalent concentrations

• Co-staining recommendations:

  • Focal adhesion markers: vinculin, paxillin

  • Actin cytoskeleton: phalloidin staining

  • For ILK at focal adhesions, consider confocal microscopy for better resolution

• Subcellular localization analysis:

  • ILK predominantly localizes to focal adhesions

  • Nuclear localization may occur under specific conditions

For dual staining, carefully select secondary antibodies to avoid cross-reactivity and consider spectral separation of fluorophores to minimize bleed-through.

How can I distinguish between different ILK isoforms in my experiments?

Distinguishing between ILK isoforms (ILK1, ILK2, ILK3) requires careful antibody selection and experimental design:

• Antibody epitope consideration:

  • Antibodies against the N-terminus of ILK1 will not detect ILK3

  • Antibodies recognizing the central part of ILK may have variable affinity for different isoforms

  • For example, an antibody directed against residues 118-241 of ILK1 will overlap with 86% of ILK3 but only 29% of ILK2

• Isoform-specific experimental design:

  • Use RT-PCR with isoform-specific primers to confirm expression at mRNA level

  • Employ size-based discrimination in high-resolution Western blots

  • Consider 2D gel electrophoresis for more definitive separation

• Functional differentiation:

  • TGF-β1 regulation appears specific to ILK2 in invasive melanoma cells, providing a functional discrimination method

  • ILK1 is ubiquitously expressed in normal tissues and upregulated in various malignancies independently of TGF-β1 stimulation

• Recombinant expression systems:

  • For definitive identification, compare against recombinant ILK isoforms expressed in appropriate systems

This differentiation is crucial since most studies focus on ILK1 without distinguishing the unique characteristics of other isoforms, potentially missing important biological functions .

What are the best approaches for studying ILK phosphorylation and kinase activity?

The kinase activity of ILK has been controversial, with recent evidence suggesting ILK is a pseudokinase . For investigating ILK phosphorylation and kinase-related functions:

• ILK phosphorylation detection:

  • Phospho-specific antibodies against key sites (Ser246, Thr173, Thr181, Ser259, Ser343)

  • Caution: validate phospho-specific antibodies using appropriate controls or phosphatase treatments

  • Use mass spectrometry for unbiased phosphorylation site identification

• Kinase activity assessment:

  • In vitro kinase assays using immunoprecipitated ILK

  • Validated substrates: MBP (myelin basic protein) and GST-PKB/Akt

  • Phosphorylation detection: [³²P]ATP labeling or phospho-specific antibodies (e.g., anti-PKB/Akt-phospho-Ser-473)

• Controls for kinase experiments:

  • Kinase-dead ILK mutants (ILK KD) as negative controls

  • Pharmacological ILK inhibitors to confirm specificity

  • Parallel analysis of known downstream targets (PKB/Akt, GSK-3)

• Pseudokinase function analysis:

  • Focus on scaffold function using protein-protein interaction studies

  • Analyze ILK's role in complexes facilitating other kinases' activity

  • Mutational analysis of the pseudoactive site, which is essential for binding to α-parvin

Recent structural and functional studies question ILK's catalytic activity, suggesting its primary role may be as a pseudokinase that mechanically couples integrin and α-parvin for mediating cell adhesion .

What methods are effective for studying ILK-protein interactions?

For comprehensive analysis of ILK protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use optimized antibodies (0.5-4.0 μg per 1-3 mg protein)

  • Validated for detecting interactions with PINCH, parvins, and integrin β subunits

  • Consider crosslinking approaches for transient interactions

  • Include appropriate negative controls (IgG isotype control)

• Proximity ligation assay (PLA):

  • Allows visualization of protein interactions in situ

  • Particularly useful for studying ILK-integrin interactions at focal adhesions

  • Requires validated antibodies from different species

• Mass spectrometry-based approaches:

  • BioID or APEX proximity labeling for identifying proximal proteins

  • Cross-linking mass spectrometry (XL-MS) for direct interaction sites

  • IP-MS for identifying ILK binding partners

• Fluorescence-based techniques:

  • FRET (Förster Resonance Energy Transfer) for direct protein interactions

  • BiFC (Bimolecular Fluorescence Complementation) for visualizing interactions in living cells

• Yeast two-hybrid screening:

  • Useful for identifying novel ILK interacting partners

  • Requires validation by secondary methods

These approaches have revealed ILK's interactions within the IPP complex and with integrin β cytoplasmic domains , which are critical for understanding its scaffold function in focal adhesions.

Why might I observe variability in ILK antibody staining patterns in different cell types?

Variability in ILK antibody staining can result from several factors:

• Differential expression of ILK isoforms:

  • Different cell types may express varying levels of ILK1, ILK2, and ILK3

  • Antibodies may have differential reactivity to these isoforms

• Post-translational modifications:

  • Phosphorylation, acetylation, ubiquitylation, SUMOylation, and methylation can affect epitope accessibility

  • Cell type-specific PTM patterns may alter antibody recognition

• Subcellular localization differences:

  • ILK primarily localizes to focal adhesions but can also be found in the nucleus and other compartments

  • Cell type-specific localization patterns may result in different staining patterns

• Cell adhesion status and ECM interactions:

  • ILK distribution changes with cell adhesion state

  • Different extracellular matrix components influence ILK localization and complex formation

• Technical variables:

  • Fixation methods affect epitope preservation differently across cell types

  • Permeabilization efficiency varies with cell membrane composition

For accurate interpretation, compare staining patterns with multiple antibodies targeting different ILK epitopes and validate localization using co-staining with known markers of focal adhesions or other relevant structures.

How can I address non-specific binding issues with ILK antibodies?

To minimize non-specific binding when using ILK antibodies:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Include 0.1-0.3% Triton X-100 in blocking buffer to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Perform antibody titration experiments to determine optimal concentration

  • Use freshly diluted antibody preparations

• Include appropriate controls:

  • Isotype controls at the same concentration as the primary antibody

  • Secondary antibody-only controls to assess background

  • Peptide competition assays to confirm specificity

• Modify washing conditions:

  • Increase number and duration of washes

  • Add detergent (0.05-0.1% Tween-20) to wash buffers

  • Consider high-salt washes (up to 500 mM NaCl) for high-background samples

• Pre-adsorption techniques:

  • Pre-adsorb antibodies with acetone powder from relevant tissues/cells

  • For tissue IHC, consider pre-adsorption with liver powder

These optimization steps help achieve cleaner results, especially important for immunohistochemistry and immunofluorescence applications where background can obscure specific signals.

What controls are essential when using ILK antibodies for functional blocking experiments?

For rigorous functional blocking experiments using anti-ILK antibodies:

An example protocol for ILK function disruption using antibodies involves adding anti-ILK antibodies (10 μg/ml) to culture medium of DRG (dorsal root ganglia) grown on laminin-1 and analyzing effects on axonal outgrowth, SCP migration, and growth cone morphology after 48 hours .

How are ILK antibodies being used in developmental and stem cell research?

ILK antibodies are valuable tools in developmental and stem cell research:

• Embryonic development studies:

  • Tracking ILK expression patterns in developing embryos

  • ILK plays crucial roles in epiblast polarization and embryonic development

  • Example: ILK is highly expressed in heart, somites, pyramidal cells, Purkinje cells, and crypt cells of intestine

• Lineage tracing and differentiation:

  • Monitoring ILK expression changes during cell differentiation

  • Co-staining with lineage-specific markers to correlate ILK with developmental stages

• Stem cell niche interactions:

  • Studying ILK's role in stem cell-matrix interactions

  • Analyzing stem cell adhesion and migration through ILK-dependent mechanisms

• Conditional knockout models:

  • ILK antibodies validate tissue-specific knockout efficiency

  • Example: ILK knockout in epiblast cells causes developmental defects

• Organoid research:

  • Investigating ILK's function in 3D organoid formation and architecture

  • Analyzing polarity establishment in epithelial organoids

Developmental researchers use ILK antibodies to understand the molecular mechanisms of cell-matrix interactions during embryogenesis and tissue morphogenesis, as ILK gene deletion leads to embryonic lethality linked to adhesive and migratory defects .

What are the latest applications of ILK antibodies in cancer research?

ILK antibodies are advancing cancer research in several key areas:

• Prognostic biomarker studies:

  • ILK upregulation has been reported in human malignancies and associated with poor prognosis

  • IHC analysis of tumor tissues with standardized scoring systems

  • Correlation with clinical outcomes and treatment responses

• Epithelial-mesenchymal transition (EMT) analysis:

  • ILK's role in EMT makes it relevant for metastasis research

  • Co-staining with EMT markers (E-cadherin, vimentin) in tumor samples

  • Analysis of ILK expression at invasive fronts

• Therapeutic target validation:

  • Antibody-mediated blocking of ILK function in cancer cells

  • Combination studies with conventional therapies

  • ILK as a potential resistance mechanism in targeted therapies

• Signaling pathway analysis:

  • Investigating ILK's role in PI3K/Akt/PKB and GSK-3 signaling in tumors

  • Correlation between ILK expression and activation of downstream targets

  • Analysis of cell cycle regulation and apoptosis resistance

• miRNA regulation studies:

  • ILK expression is suppressed by various miRNAs (miR-542-3p, miR-625, miR-145/143)

  • Therapeutic implications of miRNA-mediated ILK regulation

Recent research shows that inhibition of ILK induces G1 phase cell cycle arrest and stimulates apoptosis in PTEN-negative prostate cancer cells, suggesting ILK inhibition as a potential therapeutic approach .

How can computational approaches enhance ILK antibody design and application?

Emerging computational methods are improving ILK antibody research:

• AI-assisted antibody design:

  • Machine learning models predict optimal epitopes for ILK targeting

  • Virtual screening identifies high-affinity antibody candidates

  • Example: The Virtual Lab approach combines ESM, AlphaFold-Multimer, and Rosetta for computational antibody design

• Active learning strategies:

  • Reduce experimental costs by iteratively expanding labeled datasets

  • Improve out-of-distribution predictions for antibody-antigen binding

  • Potential to reduce required experimental samples by up to 35%

• Structure-based epitope prediction:

  • Computational identification of accessible ILK epitopes

  • Epitope conservation analysis across species for broad-reactivity antibodies

  • Integration of post-translational modification data for epitope selection

• Data mining and literature analysis:

  • Natural language processing to extract ILK-specific information from research literature

  • Meta-analysis of ILK antibody performance across published studies

• Binding affinity prediction:

  • Computational tools to estimate antibody-ILK binding parameters

  • In silico affinity maturation to enhance antibody specificity

  • Virtual docking to model antibody-ILK interactions

These computational approaches can significantly accelerate ILK antibody development by reducing experimental iterations and providing structural insights that guide rational antibody design .

What are the current controversies and unresolved questions in ILK research?

Several significant controversies and unresolved questions persist in ILK research:

• Kinase activity debate:

  • Despite initial classification as a serine/threonine-protein kinase, ILK's catalytic activity is questioned due to structural and functional issues

  • The exact molecular mechanism of signal transduction by ILK remains unsolved

  • Evidence suggests ILK functions as a pseudokinase rather than a true kinase

• Isoform-specific functions:

  • While three ILK isoforms (ILK1, ILK2, ILK3) vary in length, domains, and modification sites, most research focuses only on ILK1

  • Functional differences between ILK isoforms remain largely unexplored

  • The expression patterns and regulation of different isoforms across tissues requires clarification

• Post-translational modifications:

  • Mass spectrometry has identified numerous PTMs of ILK (phosphorylation, acetylation, ubiquitylation, SUMOylation, methylation)

  • The exact functions of these modifications remain largely unclarified

  • The regulatory mechanisms and cross-talk between different PTMs need further investigation

• Nuclear functions:

  • ILK has been detected in the nucleus where it may act as a negative regulator of transcription

  • The mechanism of nuclear shuttling and the specific nuclear roles of ILK are not fully established

• Therapeutic potential:

  • Whether targeting ILK has clinical utility remains uncertain

  • The specificity and efficacy of ILK inhibitors in disease models require further validation

Resolving these questions will require development of more specific antibodies, particularly those that can distinguish between ILK isoforms and post-translational modifications.

What new antibody technologies might advance ILK research in the future?

Emerging antibody technologies hold promise for advancing ILK research:

• Single-domain antibodies and nanobodies:

  • Smaller size allows better penetration into dense tissues and cells

  • Potential for intracellular expression to target specific ILK pools

  • Example: Computational approaches like the Virtual Lab could design nanobodies with specific binding properties to ILK domains

• Recombinant antibody engineering:

  • Production of antibodies with precisely defined binding characteristics

  • ZooMAb® technology offers exceptionally stable antibodies without animal sacrifice

  • Site-specific conjugation for advanced imaging applications

• Genetically encoded intrabodies:

  • Express antibody fragments intracellularly to visualize and manipulate ILK in living cells

  • Domain-specific targeting to disrupt specific ILK functions while preserving others

  • Fusion to degradation domains for rapid ILK depletion

• Phospho-specific and conformation-specific antibodies:

  • Detection of specific ILK activation states

  • Monitoring dynamic changes in ILK phosphorylation

  • Distinguishing between open/closed conformations of ILK

• Multiplexed antibody approaches:

  • Cyclic immunofluorescence for simultaneous detection of multiple ILK-associated proteins

  • Mass cytometry (CyTOF) for high-dimensional analysis of ILK signaling networks

  • Spatial transcriptomics combined with antibody detection

These advanced technologies will allow researchers to interrogate ILK function with unprecedented specificity and temporal resolution, potentially resolving current controversies and revealing new biological functions.