ILKAP Antibody

What is ILKAP and what is its significance in cellular signaling?

ILKAP (Integrin-linked kinase-associated serine/threonine phosphatase 2C) is a protein phosphatase that selectively associates with integrin-linked kinase (ILK) to modulate cell adhesion and growth factor signaling. It plays a critical role in:

• Regulating cell cycle progression via dephosphorylation of substrates crucial for cell proliferation

• Inhibiting the ILK-GSK3B signaling axis

• Potentially inhibiting oncogenic transformation

• Contributing to DNA repair mechanisms in p53-wildtype cells

• HIF-1α dephosphorylation under hypoxic conditions

The protein has a calculated molecular weight of 43 kDa, though it is typically observed at 43-47 kDa in experimental conditions .

What applications are ILKAP antibodies validated for?

Based on current research literature and commercial product validation data, ILKAP antibodies have been confirmed effective for:

It is recommended to titrate the antibody in each testing system to obtain optimal results, as the ideal dilution can be sample-dependent .

What are the optimal protocols for using ILKAP antibodies in Western blot applications?

For optimal Western blot results with ILKAP antibodies:

Sample Preparation:

• Use protein extracts from tissues with known ILKAP expression (human heart tissue, HepG2 cells, K-562 cells have shown positive detection)

• Prepare lysates under reducing conditions

Protocol Recommendations:

• Separate proteins on 10-12% SDS-PAGE gels

• Transfer to PVDF membrane (preferred over nitrocellulose for this protein)

• Block with 5% non-fat milk or BSA in TBST

• Dilute primary antibody (1:500-1:3000) in blocking buffer

• Incubate overnight at 4°C

• Wash 3-5 times with TBST

• Incubate with appropriate HRP-conjugated secondary antibody

• Develop using ECL detection system

Expected Results:

• ILKAP should be detected at approximately 43-47 kDa

• PC-3, HepG2, HEK293 and K-562 cell lines serve as good positive controls

What are the key experimental design considerations when studying ILKAP?

When designing experiments involving ILKAP:

• Experimental Controls:

  • Include both positive controls (HepG2, HEK293, PC-3 cells)

  • Include negative controls (tissues/cells with confirmed low ILKAP expression)

  • For antibody validation, include isotype controls in IP experiments

• Sample Selection:

  • Consider p53 status of cell lines, as ILKAP effects differ between p53-wildtype and p53-mutant cells

  • Test multiple cell types when investigating novel functions, as effects may be cell-type specific

• Experimental Variables:

  • Account for potential systematic differences between datasets when comparing multiple experiments

  • Carefully document culture conditions, as cell adhesion status affects ILKAP function

• Statistical Analysis:

  • Use appropriate statistical tests based on experimental design

  • Consider the high variability often observed in phosphatase activity assays

• Validation Methods:

  • Confirm antibody specificity using knockdown/knockout controls

  • Validate physiological relevance using multiple methodological approaches

How do monoclonal and polyclonal ILKAP antibodies differ in research applications?

The choice between monoclonal and polyclonal ILKAP antibodies should be based on your specific research needs:

Monoclonal ILKAP Antibodies:

• Recognize a single epitope on ILKAP

• Exhibit high specificity with minimal non-specific cross-reactivity

• Show minimal batch-to-batch variation

• Examples include rabbit recombinant monoclonal antibodies (clone EPR16145)

• Most suitable for: precise epitope targeting, applications requiring high reproducibility, quantitative analyses

Polyclonal ILKAP Antibodies:

• Recognize multiple epitopes on ILKAP

• May provide stronger signals by binding to several different epitopes

• Subject to higher batch-to-batch variability

• Examples include rabbit polyclonal antibodies (16017-1-AP)

• Most suitable for: detecting low-abundance targets, applications where signal amplification is needed

Recombinant Antibody Advantages:

• Long-term, secured supply with minimal batch-to-batch variation

• Known and defined antibody-encoding sequence

• Can be further engineered for specific applications

• Recommended when experimental reproducibility is critical

For ILKAP research, recombinant monoclonal antibodies are increasingly preferred due to their consistency and specificity, particularly for mechanistic studies examining ILKAP's role in signaling pathways .

How can ILKAP antibodies be used to investigate its role in cancer progression?

ILKAP has been implicated in several cancer types, and antibodies can be valuable tools for investigating its role:

Glioblastoma Multiforme (GBM) Research:

• ILKAP depletion sensitizes p53-wildtype GBM cells to radiotherapy, but not p53-mutant cells

• This is associated with inactivated GSK3β and reduced proliferation

• ILKAP knockdown leads to elevated levels of radiation-induced γH2AX/53BP1-positive foci

Methodological Approach:

• Use ILKAP antibodies to assess baseline expression in tumor vs. normal tissues

• Perform knockdown studies using siRNA/shRNA followed by Western blot validation

• Analyze proliferation, migration, and radiation sensitivity in knockdown vs. control cells

• Investigate ILKAP's interaction with known partners (ILK, PINCH1) using co-immunoprecipitation

• Assess downstream pathway activation (particularly GSK3β phosphorylation)

Melanoma Research:

• ILKAP has been implicated in susceptibility to malignant melanoma

• Altered expression patterns may correlate with disease progression

Experimental Design Recommendations:

• Compare ILKAP expression across cancer stages using tissue microarrays

• Correlate expression with clinical outcomes

• Use both IHC and WB to validate findings

• Consider p53 status of cell lines and tumors when interpreting results

What are the techniques for studying ILKAP's interaction with HIF-1α and implications for hypoxia response?

Recent research has revealed that ILKAP physically interacts with HIF-1α and induces its dephosphorylation, with implications for hypoxia-induced apoptosis . To investigate this interaction:

Co-immunoprecipitation Protocol:

• Induce hypoxic conditions (1% O₂) or use chemical inducers (CoCl₂, DFO)

• Prepare cell lysates in non-denaturing buffer containing phosphatase inhibitors

• Immunoprecipitate using anti-ILKAP or anti-HIF-1α antibodies

• Resolve by SDS-PAGE and immunoblot for the respective partner protein

• Include IgG control immunoprecipitations to verify specificity

Functional Analysis Methods:

• HIF-1α Transcriptional Activity:

  • Transfect cells with hypoxia-response element (HRE)-containing luciferase reporter

  • Manipulate ILKAP levels (overexpression or knockdown)

  • Measure luciferase activity under hypoxic conditions

• Phosphorylation Status Analysis:

  • Use phospho-specific antibodies if available

  • Alternatively, perform phosphatase treatment of immunoprecipitated HIF-1α

  • Compare migration patterns by SDS-PAGE

• Cell Viability Assessment:

  • Measure apoptosis using flow cytometry, TUNEL assay, or caspase activity

  • Compare between ILKAP-manipulated and control cells under hypoxia

  • Assess HIF-1α-p53 interaction as a downstream effect

Data Interpretation Considerations:

• The ILKAP-HIF-1α interaction appears to be critical for severe hypoxia-induced cell apoptosis

• Both gain and loss of function approaches (overexpression and shRNA) should be employed

• Results may be cell-type dependent and influenced by p53 status

What methodologies help resolve contradictory data in ILKAP phosphatase activity studies?

Researchers sometimes encounter contradictory data when studying ILKAP phosphatase activity. These methodological approaches can help resolve inconsistencies:

1. Systematic Analysis of Experimental Variables:

• Document detailed experimental conditions that may affect phosphatase activity:

  • Buffer composition (particularly Mn²⁺/Mg²⁺ concentration)

  • Substrate concentration and purity

  • Incubation time and temperature

  • Presence of phosphatase inhibitors

2. Quality Control Analysis:

• Perform quality control evaluations of data:

  • Assess control signal distributions between different studies

  • Analyze correlation patterns between datasets

  • Identify systematic differences that could be confounding factors

3. Standardization of Phosphatase Assays:

• Use recombinant proteins expressed in the same system

• Perform side-by-side comparisons with known phosphatases

• Include multiple substrate controls

4. Advanced Data Analysis Approaches:

• Apply statistical methods that equalize variance between studies

• Create differentially expressed gene (DEG) lists using consistent methodology

• Use multiple pathway analysis tools (e.g., IPA and MetaCore/MetaTox)

5. Integration of Multiple Methods:

• Combine in vitro phosphatase assays with cellular studies

• Use both gain and loss of function approaches

• Validate key findings with orthogonal techniques

Table: Common Sources of Variability in ILKAP Studies

How can researchers optimize ILKAP antibody selection for specific experimental needs?

Selecting the optimal ILKAP antibody requires careful consideration of several factors:

1. Application-Specific Selection:

• For Western blot: Both monoclonal and polyclonal antibodies work well; consider expected protein level

• For IHC: Test different antigen retrieval methods; TE buffer pH 9.0 is recommended for some antibodies

• For IP: Monoclonal antibodies often provide cleaner results with less background

• For multiple applications: Select antibodies validated across all needed applications

2. Antibody Format Considerations:

• Unconjugated: Most versatile, used with secondary detection systems

• Conjugated (HRP, fluorophores): Eliminates secondary antibody step

• Carrier-free: Required for antibody labeling or functional assays

3. Species Reactivity:

• Human ILKAP is most extensively characterized

• For cross-species studies, select antibodies validated across species (human/mouse/rat)

• Confirm reactivity in your specific sample type

4. Epitope Location:

• N-terminal vs C-terminal targeting may affect detection of splice variants or cleaved forms

• For protein interaction studies, select antibodies that don't interfere with binding regions

5. Validation Requirements:

• Review validation data specific to your application and cell/tissue type

• Examine images from manufacturer validation galleries

• Consider published literature citing specific antibody clones

6. Quality Control Considerations:

• Recombinant monoclonal antibodies offer superior batch-to-batch consistency

• For reproducible long-term studies, prioritize antibodies with documented consistency

Decision Matrix for ILKAP Antibody Selection:

What are common issues with ILKAP antibodies in Western blot and how can they be resolved?

Researchers may encounter several challenges when using ILKAP antibodies in Western blot applications:

Issue: Multiple bands or unexpected molecular weight

• Cause: Post-translational modifications, splice variants, degradation products

• Solution:

  • Compare results with multiple antibodies targeting different epitopes

  • Include positive controls with known ILKAP expression (HepG2, K-562 cells)

  • Use phosphatase treatment to determine if bands represent phosphorylated forms

  • Observe that ILKAP is typically detected at 43-47 kDa

Issue: Weak or no signal

• Cause: Low expression, inefficient transfer, suboptimal antibody concentration

• Solution:

  • Increase protein loading (start with 25-50 μg total protein)

  • Optimize antibody concentration (try 1:500 dilution first, then adjust)

  • Extend primary antibody incubation to overnight at 4°C

  • Use enhanced chemiluminescence detection systems

Issue: High background

• Cause: Insufficient blocking, excessive antibody concentration, cross-reactivity

• Solution:

  • Increase blocking time (1-2 hours) or concentration (5% BSA or milk)

  • Dilute primary antibody further

  • Increase wash steps (5 x 5 minutes with TBST)

  • Try alternative blocking reagents (BSA vs. milk)

Issue: Inconsistent results between experiments

• Cause: Variable cell culture conditions, sample preparation differences

• Solution:

  • Standardize lysate preparation (use phosphatase and protease inhibitors)

  • Control cell confluency and passage number

  • Document experimental conditions thoroughly

  • Consider using recombinant monoclonal antibodies for better consistency

How can researchers verify ILKAP antibody specificity and validate experimental findings?

Validating ILKAP antibody specificity is crucial for experimental reliability:

1. Genetic Knockdown/Knockout Controls:

• Perform siRNA or shRNA knockdown of ILKAP

• Compare antibody reactivity in control vs. knockdown samples

• The signal should decrease proportionally to knockdown efficiency

2. Peptide Competition Assays:

• Pre-incubate antibody with excess immunizing peptide

• The specific signal should be blocked or significantly reduced

• Non-specific signals will remain unchanged

3. Multiple Antibody Validation:

• Test different antibodies targeting distinct ILKAP epitopes

• Consistent results across antibodies increase confidence

• Compare monoclonal and polyclonal antibodies for complementary information

4. Positive Control Tissues/Cells:

• Human heart tissue, HepG2, PC-3, K-562, and HEK293 cells show reliable ILKAP expression

• Include these as positive controls in validation experiments

5. Orthogonal Validation Methods:

• Complement protein detection with mRNA analysis (RT-PCR, RNA-seq)

• Verify subcellular localization using fractionation and immunofluorescence

• Confirm protein interactions using multiple approaches (co-IP, proximity ligation assay)

6. Reproducibility Assessment:

• Test batch-to-batch consistency of antibodies

• Document all experimental conditions thoroughly

• Consider using recombinant antibodies for critical experiments

What are emerging applications of ILKAP antibodies in cancer research?

Recent findings have opened new avenues for ILKAP antibody applications in cancer research:

Radioresistance in Glioblastoma:

• ILKAP depletion sensitizes p53-wildtype GBM cells to radiotherapy

• This effect is associated with elevated levels of radiation-induced γH2AX/53BP1-positive foci

• ILKAP antibodies can be used to correlate expression with treatment response

Methodological approach:

• Stratify patient samples by ILKAP expression using IHC

• Correlate with radiotherapy response and survival outcomes

• Develop predictive biomarker panels including ILKAP

Melanoma Susceptibility:

• ILKAP has been implicated in susceptibility to malignant melanoma

• Antibodies can help characterize expression patterns across melanoma progression

Experimental approach:

• Compare ILKAP expression in normal melanocytes, benign nevi, and melanoma stages

• Correlate with clinical and pathological parameters

• Investigate mechanism through pathway analysis

Hypoxia Response Modulation:

• ILKAP binds to and dephosphorylates HIF-1α in hypoxic conditions

• This interaction is essential for hypoxia-induced apoptosis

• ILKAP antibodies can help elucidate this regulatory mechanism

Future Research Directions:

• Development of phospho-specific ILKAP antibodies to study its regulation

• Use of ILKAP antibodies to identify novel interaction partners through immunoprecipitation-mass spectrometry

• Application in patient stratification for personalized treatment approaches

How can ILKAP antibodies be employed in studies of drug resistance mechanisms?

Research on drug resistance mechanisms can benefit from ILKAP antibodies in several ways:

Investigation of Signaling Pathway Modulation:

• ILKAP inhibits the ILK-GSK3B signaling axis

• This pathway is implicated in drug resistance in multiple cancers

• ILKAP antibodies can help monitor pathway activity during drug treatment

Methodological approach:

• Monitor ILKAP expression before and after drug treatment

• Correlate with downstream pathway activation (GSK3B phosphorylation)

• Compare sensitive vs. resistant cell populations

ILKAP in T-ALL Drug Resistance Models:

• While not directly focused on ILKAP, studies on T-ALL drug resistance models demonstrate how protein expression patterns relate to resistance

• Similar approaches can be applied to study ILKAP's potential role in drug response

Experimental design considerations:

• Establish drug-resistant cell lines through gradual exposure

• Compare ILKAP expression and activity between parental and resistant lines

• Manipulate ILKAP levels to assess impact on drug sensitivity

• Use antibodies to track protein interactions and pathway activation

Translational Research Applications:

• Develop tissue microarrays of patient samples before and after treatment

• Use ILKAP antibodies to assess expression changes

• Correlate with treatment response and progression-free survival

Future Directions:

• Development of patient-derived xenograft models to study ILKAP's role in drug resistance in vivo

• Integration of ILKAP expression data with other biomarkers to create predictive signatures

• Investigation of ILKAP as a potential therapeutic target to overcome resistance