IL24 Antibody

What is IL-24 and why is it significant in immunological research?

IL-24 (Interleukin-24), also known as MDA-7 (melanoma differentiation-associated gene-7) or ST16, belongs to the IL-10 family of helical cytokines. It has gained significant research attention due to its:

• Antiproliferative properties, particularly on melanoma cells

• Role in terminal cell differentiation

• Tumor-suppressive functions independent of classical cytokine activity

• Involvement in both innate and adaptive immune responses

IL-24 functions through two heterodimeric receptor complexes: IL-20Rα/IL-20Rβ (shared with IL-19 and IL-20) and IL-22R/IL-20Rβ (shared with IL-20), activating signal transduction pathways including STAT3 .

What are the molecular characteristics of IL-24 protein that researchers should consider when selecting antibodies?

When selecting IL-24 antibodies, researchers should account for several key molecular features:

• Protein Structure: Mature human IL-24 consists of 158 amino acids (aa) with a precursor form containing a 48 aa signal sequence

• Molecular Weight: The calculated molecular weight is approximately 24 kDa, but observed weights typically range from 25-35 kDa due to post-translational modifications, particularly glycosylation

• Species Homology: Human IL-24 shares 69% amino acid sequence identity with mouse/rat variants, allowing human IL-24 to be active in rodent systems

• Glycosylation Status: Contains three potential N-linked glycosylation sites, with glycosylation believed to be essential for biological activity

• Oligomerization: Can exist as either monomer or dimer when secreted

These characteristics influence epitope availability and antibody binding efficiency in different applications.

How should researchers approach validation of IL-24 antibodies for their specific experimental systems?

Proper validation should include:

• Cross-reactivity assessment: Verify reactivity with target species. For example, antibody products 26772-1-AP and 12064-1-AP show different species reactivity profiles (26772-1-AP reacts with human and mouse, while 12064-1-AP is human-specific)

• Application-specific validation:

  • For Western blot: Confirm detection at expected molecular weight (24-35 kDa range) using positive controls

  • For IHC: Test on known IL-24-expressing tissues (e.g., human colon, malignant melanoma)

  • Include knockout/knockdown controls where possible

• Dilution optimization: Test recommended dilution ranges (e.g., 1:500-1:1000 for WB, 1:800-1:3200 for IHC with antibody 26772-1-AP)

• Buffer compatibility: Confirm compatibility with sample preparation methods and buffer systems

• Reproducibility testing: Ensure consistent results across multiple experiments and sample types

What are the optimal protocols for using IL-24 antibodies in immunohistochemistry applications?

For optimal IHC results with IL-24 antibodies:

Sample Preparation and Antigen Retrieval:

• Fixed tissue sections (FFPE) require appropriate antigen retrieval

• For antibodies like 26772-1-AP and 12064-1-AP, use TE buffer pH 9.0 as primary recommendation

• Alternative: citrate buffer pH 6.0 may also be effective

Protocol Optimization:

• Deparaffinize and rehydrate tissue sections using standard procedures

• Perform antigen retrieval with recommended buffer system

• Block endogenous peroxidase (for chromogenic detection) and non-specific binding sites

• Apply primary IL-24 antibody at recommended dilution:

  • For 26772-1-AP: 1:800-1:3200

  • For 12064-1-AP: 1:50-1:500

• Incubate at 4°C overnight (or per manufacturer's recommendation)

• Apply appropriate detection system and counterstain

Tissue-Specific Considerations:

• Human colon tissue and malignant melanoma are positive controls for many IL-24 antibodies

• Antibody 12064-1-AP shows positive reactivity in multiple cancer tissues (prostate, colon, and lung cancer tissues)

Result Interpretation:

• Compare staining patterns with literature data

• Cellular localization should align with IL-24's reported distribution (cytoplasmic and secreted)

How should researchers optimize Western blot protocols for detecting IL-24 protein?

For successful Western blot detection of IL-24:

Sample Preparation:

• Mouse spleen tissue is an appropriate positive control for antibody 26772-1-AP

• For recombinant protein controls, use human IL-24 with verified identity

Protocol Optimization:

• Use appropriate lysis buffer with protease inhibitors

• Load 20-40 μg of total protein per lane

• Use 12-15% acrylamide gels for better resolution of the 24-35 kDa range

• Transfer to PVDF or nitrocellulose membrane using standard protocols

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

• Apply IL-24 antibody at optimized dilution:

  • For 26772-1-AP: 1:500-1:1000

  • For 12064-1-AP: 1:1000-1:4000

• Incubate overnight at 4°C

• Wash and apply appropriate secondary antibody

• Develop using enhanced chemiluminescence

Troubleshooting Considerations:

• For glycosylated IL-24 detection (35-40 kDa), ensure your gel percentage and running conditions allow visualization of this range

• Multiple bands may represent different glycosylation states or proteolytic fragments

• Consider deglycosylation treatments if analyzing core protein size

What considerations are important when designing IL-24 knockdown/knockout validation experiments?

When designing IL-24 knockdown/knockout experiments:

Validation Approaches:

• siRNA/shRNA: For temporary knockdown, design targeting multiple regions of IL-24 mRNA

• CRISPR-Cas9: For permanent knockout, target early exons to ensure complete protein disruption

• Validation Controls: Include scrambled siRNA/shRNA or non-targeting gRNA controls

Functional Validation:

• Measure IL-24 expression by qPCR and Western blot

• Assess key downstream pathways (e.g., JAK/STAT signaling)

• Examine phenotypic changes in IL-24-dependent processes:

  • For Th9 cells: Monitor proliferation and survival as Il24−/− Th9 cells show increased proliferation but decreased survival in vitro

  • For tumor models: Assess cytotoxicity and anti-tumor responses

Published Validation Examples:

• Study of IL-24 in Th9 cells used Il24−/− mice and demonstrated that knockout affected tumor-specific cytotoxicity and IL-9 production

• IL-24 receptor (IL-20R2) blocking antibody has been used as an alternative approach to disrupt IL-24 signaling

How can IL-24 antibodies be utilized to investigate the role of IL-24 in cancer immunotherapy research?

IL-24 antibodies serve multiple functions in cancer immunotherapy research:

Mechanistic Studies:

• Characterize IL-24 expression in tumor vs. normal tissues using IHC

• Monitor changes in IL-24 levels following immunotherapeutic interventions

• Track IL-24-producing cells within the tumor microenvironment

Therapeutic Development Applications:

• Assess IL-24's anti-tumor activity: IL-24 functions as a powerful antitumor effector for Th9 cells

• Investigate combination approaches: IL-24 sensitizes cancer cells to TLR3-mediated apoptosis, particularly relevant for viral-based immunotherapies

• Monitor receptor expression (IL-20R1/IL-20R2/IL-22R) in responding vs. non-responding tumors

Experimental Models:

• In adoptive T-cell therapy models, IL-24 antibodies can track transferred Th9 cells and correlate IL-24 expression with therapeutic efficacy

• For lung metastasis models, immunotherapy with Il24 knockout Th9 cells showed less tumor inhibition than wild-type Th9 cells, highlighting IL-24's central role

Functional Assays:

• Cytotoxicity assays show IL-24 knockout decreases tumor-specific killing by Th9 cells

• IL-24 enables formation of a TLR3-associated death-inducing signaling complex that enhances apoptosis when combined with TLR3 agonists like poly(I:C)

What methodologies are recommended for studying IL-24's role in T-cell biology and inflammatory diseases?

For investigating IL-24 in T-cell biology:

Isolation and Culture Techniques:

• For Th9 polarization: Culture naive CD4+ T cells with TGF-β and IL-4, monitoring IL-24 expression alongside IL-9

• Use flow cytometry with IL-24 antibodies to identify IL-24-producing T-cell subsets

Functional Assays:

• Cell proliferation: Il24−/− Th9 cells show increased proliferation compared to wild-type Th9 cells

• Cell survival analysis: Il24−/− Th9 cells exhibit higher apoptosis rates in extended cultures

• In vivo tracking: CFSE-labeled Th9 cells from WT vs. Il24−/− mice show differential persistence in adoptive transfer models

Inflammatory Disease Models:

• In autoimmune diseases: IL-24 may suppress pathogenic Th17 responses by regulating IL-17F and GM-CSF production

• In infectious diseases: IL-24 limits IFN-γ and IL-17A from Th1/Tc1 and Th17/Tc17 cells in patients with lymphatic filariasis and tuberculosis

Methodological Table for T-cell Studies:

How can researchers investigate the therapeutic potential of IL-24 in liver diseases?

For studying IL-24 in liver diseases:

Experimental Models:

• Thioacetamide (TAA)-induced acute and chronic liver injury models provide a platform to test IL-24's protective effects

• Can be applied as preventive (pre-treatment) or therapeutic intervention

Administration Protocols:

• Preventive approach: recombinant mouse IL-24 protein (1 mg/kg) intraperitoneally 24h before TAA treatment, followed by two additional injections at 24h and 48h post-TAA

• Therapeutic approach: recombinant mouse IL-24 protein (1.5 mg/kg) at 24h and 48h after TAA treatment

Assessment Parameters:

• Liver damage markers: ALT/AST, histopathological analysis

• Inflammation markers: pro-inflammatory cytokines, immune cell infiltration

• Fibrosis markers: collagen deposition, activation of hepatic stellate cells

• Oxidative stress parameters

Translational Relevance:

• Clinical correlation: Lower IL-24/IL-20 ratio is observed in patients with more severe liver fibrosis

• IL-24 protects against liver fibrosis by blocking hepatic stellate cell activation and proliferation

• Therapeutic potential for treating various types of liver injuries

How should researchers interpret discrepancies in IL-24 protein molecular weight across different detection methods?

When interpreting IL-24 molecular weight variations:

Expected Size Range:

• Calculated molecular weight: 24 kDa for the mature protein (207 aa with 48 aa signal sequence removed)

• Observed molecular weight range: 25-35 kDa on Western blots

• In some systems, IL-24 appears as a 35-40 kDa phosphorylated glycoprotein

Sources of Variation:

• Post-translational modifications: IL-24 contains three potential N-linked glycosylation sites

• Oligomerization: Can exist as monomer or dimer in secreted form

• Tissue-specific processing: Different cell types may process IL-24 differently

• Experimental conditions: Sample preparation methods can affect observed size

Verification Approaches:

• Enzymatic deglycosylation to confirm contribution of glycosylation to size differences

• Reducing vs. non-reducing conditions to assess dimerization

• Phosphatase treatment to evaluate phosphorylation contribution

• Comparison with recombinant IL-24 standards of known modifications

What strategies can address non-specific binding issues when using IL-24 antibodies in complex biological samples?

To minimize non-specific binding:

Optimization Strategies:

• Blocking optimization:

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

  • Extend blocking time for high-background samples

• Antibody dilution refinement:

  • Titrate antibodies beyond manufacturer's recommended range

  • For 26772-1-AP: Test narrower ranges within 1:800-1:3200 for IHC

  • For 12064-1-AP: Evaluate dilutions between 1:50-1:500 for IHC

• Buffer modifications:

  • Increase detergent concentration (0.1-0.3% Tween-20) in wash buffers

  • Add low concentrations of competing proteins to reduce non-specific interactions

• Sample preparation optimization:

  • More rigorous antigen retrieval for IHC (test both citrate pH 6.0 and TE pH 9.0)

  • More thorough membrane blocking for Western blot

• Control inclusion:

  • Use IL-24 knockout/knockdown samples as negative controls

  • Pre-adsorption of antibody with recombinant IL-24 to confirm specificity

How can researchers reconcile conflicting findings about IL-24's immunomodulatory effects in different disease contexts?

To address conflicting findings about IL-24:

Context-Dependent Effects:

• IL-24 exhibits seemingly opposing effects in different disease models:

  • In some infectious diseases, IL-24 limits IFN-γ and IL-17A production from T cells

  • In colorectal adenocarcinoma, IL-24 promotes T cell activation with elevated IFN-γ and IL-17A expression

  • In Th17-mediated experimental autoimmune uveitis, IL-24 suppresses pathogenic Th17 response

Reconciliation Approaches:

• Cell-type specific analysis: Determine effects on specific immune cell subsets rather than total tissue or mixed populations

• Concentration dependency: IL-24 may have dose-dependent effects

  • Low concentrations: Induces type I proinflammatory cytokines (IFN-γ, IL-1β, IL-12, TNF-α)

  • High concentrations: Strong inducer of apoptosis in tumor cells but not normal cells

• Receptor expression profiling: Determine the expression patterns of IL-24 receptor complexes (IL-20Rα/IL-20Rβ vs. IL-22R/IL-20Rβ) in different cell types

• Temporal dynamics: Monitor effects over time, as IL-24's role may shift during disease progression

  • Example: IL-24 induction by oncogenes may support tumor growth at early stages but inhibit progression later

• Pathway analysis: Determine which signaling pathways are activated in different contexts

  • IL-24 can activate STAT3 in endothelial cells, blocking differentiation

  • In Th17 cells, IL-24 expression is induced by IL-17A through NFκB signaling in an autocrine manner

What are the latest developments in utilizing IL-24 antibodies for cancer therapeutic applications?

Recent research highlights several innovative applications:

Cancer Immunotherapy Enhancement:

• IL-24 identified as a key effector of Th9 cell-mediated tumor immunotherapy, with Il24-knockout Th9 cells showing reduced therapeutic efficacy

• Combination approaches using IL-24 with TLR3 agonists (poly(I:C)) show enhanced tumor cell apoptosis through formation of an atypical death-inducing signaling complex

Mechanism-Based Applications:

• IL-24 antibodies can monitor target engagement in experimental therapeutics

• Detection of IL-24 receptor complexes (IL-20Rα/IL-20Rβ or IL-22R/IL-20Rβ) may predict therapeutic response

• Monitoring IL-24-mediated signaling (STAT3 activation) correlates with anti-angiogenic effects

Emerging Biomarker Applications:

• Expression levels of IL-24 in tumor tissue may serve as prognostic indicators

• IL-24/IL-20 ratio shows potential as a biomarker for disease severity in liver fibrosis

How can researchers investigate the interplay between IL-24 and other cytokines in complex immunological networks?

To study IL-24 within cytokine networks:

Experimental Approaches:

Network Analysis Table:

This methodological approach helps decipher the complex interplay between IL-24 and other cytokines in different immunological contexts.