IFI30 Antibody

What is IFI30 and why is it important in biological research?

IFI30, also known as gamma-interferon-inducible lysosomal thiol reductase (GILT), is a protein that plays critical roles in various biological processes. In humans, the canonical protein consists of 250 amino acid residues with a molecular mass of approximately 28 kDa . It localizes primarily to lysosomes and is also secreted . IFI30 is important in research because it:

• Enhances MHC class II-restricted antigen processing, essential for CD4+ T lymphocyte activation

• Participates in antibacterial infection responses

• Contributes to reactive oxygen species production

• Induces autophagy

• Inhibits the entry of selected enveloped RNA viruses

The protein has gained significant research interest due to its aberrant expression in several cancer types, including diffuse large B-cell lymphoma (DLBCL), melanoma, breast cancer, and glioma .

What are the key structural and biochemical properties of IFI30?

IFI30 is a member of the GILT protein family and is characterized by several important biochemical and structural features:

• Molecular weight: Calculated at 29 kDa based on 261 amino acids; observed at 29 kDa in experimental conditions

• Post-translational modifications: Undergoes N-glycosylation and protein cleavage

• Subcellular localization: Primarily found in lysosomes and is also secreted into the extracellular space

• Function: Involved in protein stabilization

• Expression pattern: Widely expressed across many human tissue types

• Orthologs: Present in mouse, rat, bovine, frog, chimpanzee, and chicken species

What are the most common applications for IFI30 antibodies in research?

IFI30 antibodies are versatile tools in molecular and cellular research, with applications including:

These applications enable researchers to study IFI30 expression, localization, and function in various experimental models .

How should I optimize IFI30 antibody dilutions for different experimental techniques?

Optimization of antibody dilution is critical for obtaining reliable and reproducible results in IFI30 research. The following methodological approach is recommended:

• For Western Blot applications:

  • Begin with a moderate dilution (1:5000) within the recommended range (1:2000-1:12000)

  • Perform a titration experiment with serial dilutions if signal strength is suboptimal

  • Consider cell/tissue type specificity, as observed in COLO 320 cells

  • Validate specificity with appropriate positive and negative controls

• For Immunohistochemistry:

  • Start with 1:4000 dilution within the recommended range (1:2000-1:8000)

  • Important antigen retrieval note: Use TE buffer pH 9.0 as the primary method, with citrate buffer pH 6.0 as an alternative

  • Tissue-specific optimization may be required, particularly for human liver cancer tissue

• For Immunofluorescence/ICC:

  • Begin with higher antibody concentration (1:200) within the recommended range (1:50-1:500)

  • Adjust based on signal-to-noise ratio in your specific cell type

  • Validated in HeLa cells, but verification in your model system is advised

For all applications, titration experiments with different antibody concentrations are essential to determine optimal conditions for your specific experimental system.

What are the recommended sample preparation methods for detecting IFI30 in different tissue types?

The detection of IFI30 requires specific sample preparation methods depending on the tissue type and analytical technique:

For protein extraction and Western blot analysis:

• Use standard lysis buffers containing protease inhibitors

• Sample types successfully used include COLO 320 cells

• Expected molecular weight on gels: 29 kDa

For tissue section preparation and IHC:

• Fixation: Standard formalin fixation and paraffin embedding

• Antigen retrieval: Primary recommendation is TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0 if primary method yields suboptimal results

• Successfully tested on human liver cancer tissue

For immunofluorescence sample preparation:

• Fixation: Standard 4% paraformaldehyde

• Permeabilization: 0.1-0.5% Triton X-100

• Blocking: 1-5% BSA or serum appropriate to secondary antibody species

• Successfully implemented in HeLa cells

Each preparation method should be optimized for the specific research question and tissue type under investigation.

How can IFI30 antibodies be utilized to study the protein's role in cancer progression and therapy resistance?

IFI30 has emerged as a significant factor in cancer biology, particularly in relation to tumor progression and therapy resistance. IFI30 antibodies can be employed in several sophisticated research applications:

• Analysis of epithelial-mesenchymal transition (EMT) processes:

  • IFI30 promotes the EMT-like phenotype in glioma cells by activating the EGFR/AKT/GSK3β/β-catenin pathway

  • Use IFI30 antibodies in co-immunoprecipitation experiments to identify protein-protein interactions within this pathway

  • Combine with antibodies against EMT markers (E-cadherin, vimentin, Slug) for multiplexed immunofluorescence to visualize transition states

• Investigation of chemoresistance mechanisms:

  • IFI30 regulates chemoresistance to temozolomide (TMZ) in glioma cells

  • Expression levels increase in response to TMZ treatment in a dose-dependent manner

  • Design experiments comparing IFI30 expression levels in parental versus TMZ-resistant cell lines using quantitative Western blot analysis

  • Correlate with patient response data using tissue microarrays and IHC

• Translational research applications:

  • Prognostic marker development: High IFI30 expression correlates with poor patient outcomes in glioblastoma

  • Target identification: IFI30 represents a potential therapeutic target for TMZ-resistant glioma

  • Patient stratification: Consider IFI30 expression levels when designing clinical trials for glioma therapies

What experimental approaches can effectively assess the functional impact of IFI30 in cellular models?

To determine the functional significance of IFI30, researchers can implement several sophisticated experimental strategies:

• Gene modulation techniques:

  • Knockdown approaches: shRNA targeting IFI30 has been effective in GSC464 cells, resulting in decreased tumor volume and weight in xenograft models

  • Overexpression studies: Transfection of IFI30 expression constructs enhances mesenchymal characteristics in glioma cell lines

  • CRISPR/Cas9 genome editing for complete knockout studies

• Functional assays for cancer-related phenotypes:

  • Cell viability: CCK-8 assays reveal decreased viability after IFI30 knockdown

  • Colony formation: Quantification of colony-forming capacity after IFI30 modulation

  • Cell migration: Wound healing and transwell assays demonstrate reduced migration following IFI30 silencing

  • Limiting dilution analysis: Evaluates self-renewal capacity of cancer stem cells with altered IFI30 expression

• Pathway analysis and signaling studies:

  • Western blot analysis of downstream effectors following IFI30 modulation, focusing on:

    • EGFR/AKT/GSK3β/β-catenin pathway components

    • EMT-related transcription factors (particularly Slug)

  • Nuclear/cytoplasmic fractionation to assess β-catenin translocation regulated by IFI30

  • Small molecule inhibitor studies (e.g., AG-1478 for EGFR inhibition) to validate pathway connections

How can researchers address common technical challenges when using IFI30 antibodies?

When working with IFI30 antibodies, researchers frequently encounter several technical challenges. Here are methodological approaches to address these issues:

• Specificity validation concerns:

  • Conduct parallel experiments with multiple antibody clones targeting different epitopes

  • Include appropriate controls: positive control (tissue/cells known to express IFI30), negative control (IFI30 knockout/knockdown samples)

  • Verify specificity with IP-Western blot confirmation

  • Consider pre-adsorption tests with immunizing peptide when available

• Background signal in immunohistochemistry:

  • Optimize blocking conditions: Increase blocking time or concentration (5% BSA or normal serum)

  • Adjust antibody concentration: Start with higher dilutions (1:8000) and titrate as needed

  • Modify antigen retrieval: Switch between recommended TE buffer pH 9.0 and alternative citrate buffer pH 6.0

  • Reduce secondary antibody concentration or use more specific detection systems

• Variable detection across different sample types:

  • Tissue-specific optimization may be required as IFI30 is widely expressed in many tissues

  • Adjust protein extraction protocols based on tissue type (e.g., brain tissue vs. cultured cells)

  • Consider post-translational modifications: IFI30 undergoes N-glycosylation and cleavage that may affect detection

How should researchers interpret conflicting data regarding IFI30 expression patterns in different experimental contexts?

Interpreting conflicting IFI30 expression data requires careful consideration of multiple factors:

• Cellular context considerations:

  • IFI30 expression varies significantly between normal and cancerous tissues

  • Expression increases with WHO grade in gliomas, with highest levels in glioblastoma multiforme (GBM)

  • IDH mutation status affects expression patterns in glioma

  • Mesenchymal glioma subtype shows particularly high IFI30 expression compared to other subtypes

• Methodological factors affecting expression analysis:

  • RNA vs. protein detection: IFI30 mRNA levels (qPCR, RNA-seq) may not directly correlate with protein expression (Western blot, IHC)

  • Antibody selection: Different antibody clones may have varying affinities for different IFI30 isoforms or post-translationally modified forms

  • Sample preparation: Fixation methods for IHC can affect epitope accessibility

• Experimental intervention effects:

  • Treatment-induced changes: TMZ treatment upregulates IFI30 expression in glioma cells

  • Temporal dynamics: Consider time-course experiments as IFI30 expression may change during disease progression

  • Microenvironmental influences: IFI30 is interferon-inducible, so inflammatory conditions in the tumor microenvironment may influence expression levels

When faced with conflicting data, a comprehensive approach involving multiple detection methods, careful consideration of experimental conditions, and validation across independent sample sets is recommended.

What are emerging applications of IFI30 antibodies in cancer research and potential therapeutic developments?

Based on current knowledge, several promising research directions for IFI30 antibodies are emerging:

• Biomarker development for cancer prognosis:

  • IFI30 expression correlates with patient survival in glioblastoma

  • Development of standardized IHC protocols for clinical implementation

  • Integration with other molecular markers for improved prognostic accuracy

  • Potential for liquid biopsy applications detecting IFI30 in extracellular vesicles

• Therapeutic resistance mechanisms:

  • IFI30 upregulation occurs in response to temozolomide treatment

  • Antibody-based detection of IFI30 could identify patients likely to develop resistance

  • Targeting the EGFR/AKT/GSK3β/β-catenin pathway influenced by IFI30 may overcome resistance

  • Combination therapy approaches based on IFI30 expression patterns

• Immunotherapy connections:

  • Given IFI30's role in MHC class II-restricted antigen processing

  • Investigation of correlations between IFI30 expression and immunotherapy response

  • Potential for enhancing immune recognition of tumors by modulating IFI30 function

  • Development of novel immunotherapeutic approaches targeting IFI30-related pathways

How can multiparametric analysis incorporating IFI30 antibodies enhance cancer classification and treatment selection?

Advanced multiparametric approaches incorporating IFI30 antibodies offer significant potential for cancer research:

• Integrated molecular classification systems:

  • Combination of IFI30 with other molecular markers for refined tumor classification

  • Integration with IDH mutation status in glioma classification schemes

  • Correlation with mesenchymal subtype identification in glioblastoma

  • Development of antibody panels for comprehensive tumor profiling

• Spatial analysis of tumor heterogeneity:

  • Multiplex immunofluorescence incorporating IFI30 antibodies with EMT markers

  • Spatial transcriptomics combined with IFI30 protein detection

  • Analysis of IFI30 expression in relation to tumor microenvironment components

  • Correlation with invasive tumor fronts vs. tumor core regions

• Therapeutic decision support:

  • IFI30 expression as a determinant for EGFR-targeted therapy selection

  • Predictive biomarker development for temozolomide response in glioma patients

  • Patient stratification for clinical trials based on IFI30-related pathway activation

  • Personalized medicine approaches incorporating IFI30 status

These emerging applications highlight the importance of incorporating IFI30 antibodies into comprehensive research strategies aimed at improving cancer classification, treatment selection, and patient outcomes.