ISG15 Antibody

What is ISG15 and why is it significant in research?

ISG15 is a 165 amino acid ubiquitin-like modifier with a molecular weight of approximately 18 kDa. It plays a key role in the innate immune response to viral infection through two mechanisms: conjugation to target proteins (ISGylation) or as a free/unconjugated protein . ISG15 consists of two tandem ubiquitin-like domains that share structural similarity with ubiquitin and other modifiers including NEDD8 and SUMO1 .

The significance of ISG15 in research stems from its critical functions in:

• Antiviral immunity against both DNA and RNA viruses, including influenza A, HIV-1, and Ebola virus

• Regulation of immune cell functions and inflammatory pathways

• Potential role as a biomarker in conditions like dermatomyositis

• Complex interactions with multiple cellular proteins and pathways

Research on ISG15 provides insights into immune regulation, viral pathogenesis, and potential therapeutic targets for infectious and autoimmune diseases.

What types of ISG15 antibodies are available for research applications?

Several types of ISG15 antibodies are available for research purposes:

The choice between polyclonal and monoclonal antibodies depends on your specific experimental needs. Polyclonal antibodies recognize multiple epitopes and may provide stronger signals, while monoclonal antibodies offer higher specificity for a single epitope .

How should I select the appropriate ISG15 antibody for my specific research application?

Selection of the appropriate ISG15 antibody should be guided by several critical factors:

• Experimental application: Verify validation data for your specific application (WB, IHC, IF, IP, etc.). For example, antibody 15981-1-AP has been validated for WB, IHC, IF/ICC, IF-P, IP, Co-IP, and RIP applications .

• Species reactivity: Ensure compatibility with your experimental model. Many antibodies react with human and mouse ISG15, but cross-reactivity with other species varies .

• Recognition of free vs. conjugated ISG15: Some antibodies specialize in detecting ISGylated proteins. For example, R&D Systems antibody A-830 specifically recovers ISGylated proteins in immunoprecipitation applications .

• Validation data: Review published literature using the antibody. For instance, antibody 15981-1-AP has been cited in 55 publications for Western blotting applications .

• Epitope location: Consider whether the antibody recognizes N-terminal, C-terminal, or internal epitopes, which may affect detection of ISG15 conjugates.

Request validation data from manufacturers to ensure the antibody performs reliably in your specific experimental system before making your selection.

What are the optimal protocols for detecting ISG15 and ISGylated proteins by Western blotting?

For optimal Western blot detection of ISG15 and ISGylated proteins:

Sample Preparation:

• Include positive controls such as IFN-α/β-treated cells, which significantly upregulate ISG15 expression

• For studying ISGylated proteins, treat cells with interferon gamma as demonstrated in the R&D Systems data

• Use appropriate lysis buffers containing protease inhibitors to prevent degradation

Protocol Optimization:

• Gel selection: Use 12-15% SDS-PAGE gels for detecting free ISG15 (15-17 kDa)

• Transfer conditions: Optimize for small proteins (free ISG15) or use gradient gels for ISGylated proteins

• Antibody dilution: Most ISG15 antibodies work effectively at 1:1000-1:4000 dilution for WB

• Detection method: Use enhanced chemiluminescence or fluorescent secondary antibodies

Expected Results:

• Free ISG15 appears as a band at 15-17 kDa

• ISGylated proteins appear as higher molecular weight bands or smears

• Research data shows significantly increased ISG15 expression in interferon-treated cells compared to untreated controls

When analyzing ISGylation patterns, compare treated vs. untreated samples to distinguish between basal and induced ISGylation.

What approaches are recommended for studying ISGylation in cellular models?

Studying ISGylation in cellular models requires specialized approaches:

Cellular Model Selection:

• A549, HeLa, and HepG2 cells are commonly used models that respond well to interferon stimulation

• Primary cells may provide more physiologically relevant insights than immortalized cell lines

Experimental Approaches:

• Interferon stimulation: Treat cells with IFN-α/β to induce ISG15 expression and ISGylation

• Expression systems: Transfect cells with HA-tagged or FLAG-tagged ISG15 constructs for easier detection

• Immunoprecipitation: Use ISG15 antibodies to pull down ISGylated proteins, followed by Western blotting

  • R&D Systems data demonstrates successful IP of ISGylated proteins using their A-830 antibody from interferon-treated A549 cells

• Proteomic analysis: Identify ISGylated proteins using mass spectrometry after immunoprecipitation

Validation Methods:

• Include controls such as cells expressing catalytically inactive ISG15 or cells treated with ISGylation inhibitors

• Verify results using siRNA knockdown of key ISGylation enzymes (UBE1L, UbcH8, HERC5)

• Use UBP43/USP18 overexpression to confirm the specificity of detected ISGylated proteins

These approaches provide complementary data to build a comprehensive understanding of ISGylation dynamics in your experimental system.

How can I effectively use ISG15 antibodies in immunohistochemistry and immunofluorescence applications?

For optimal results with ISG15 antibodies in IHC/IF applications:

Sample Preparation:

• For IHC-P: Use appropriate fixation and antigen retrieval methods. Proteintech recommends TE buffer pH 9.0 for antigen retrieval with their antibody

• For IF: Fix cells with 4% paraformaldehyde followed by permeabilization with 0.1-0.5% Triton X-100

Protocol Optimization:

• Antibody dilution: Use 1:50-1:500 dilution for both IHC and IF applications with most ISG15 antibodies

• Blocking: Block with 5-10% normal serum from the species of the secondary antibody

• Incubation conditions: Incubate primary antibody overnight at 4°C for optimal binding

• Secondary antibody: Use fluorophore-conjugated secondary antibodies appropriate for your imaging system

Expected Patterns:

• In dermatomyositis tissue samples, ISG15 expression is universally positive, particularly in perivascular regions and lymphocytes

• Control tissues typically show negative expression for ISG15

• Subcellular localization may include both cytoplasmic and nuclear patterns depending on the cellular context

Including both positive controls (e.g., interferon-stimulated tissues) and negative controls is essential for confirming specificity.

Why might I observe multiple bands or unexpected patterns when using ISG15 antibodies in Western blot?

Multiple bands or unexpected patterns when using ISG15 antibodies may result from several factors:

Common Causes and Solutions:

• ISGylated proteins: Higher molecular weight bands often represent ISG15-conjugated proteins, especially in interferon-stimulated samples. This is expected and represents biologically relevant modifications .

  • Solution: Include non-stimulated controls to distinguish between basal and induced ISGylation patterns

• Protein degradation: Additional lower molecular weight bands may indicate degradation.

  • Solution: Use fresh samples with protease inhibitors and maintain cold conditions during preparation

• Cross-reactivity: Some antibodies may cross-react with other ubiquitin-like modifiers due to structural similarities.

  • Solution: Validate specificity using ISG15 knockout/knockdown controls or blocking peptides

• Post-translational modifications: ISG15 itself may undergo modifications affecting its mobility.

  • Solution: Include appropriate controls such as recombinant ISG15 protein

• Antibody specificity: Different antibody clones recognize different epitopes, potentially resulting in varying banding patterns.

  • Solution: Compare results with multiple antibodies targeting different regions of ISG15

The observed molecular weight of ISG15 typically ranges from 15-17 kDa, as noted in the Proteintech antibody data , but conjugated forms will appear at higher molecular weights.

What controls should I include when performing ISG15 detection experiments?

Robust ISG15 detection experiments require these essential controls:

Positive Controls:

• Interferon-treated samples: Cells treated with type I interferons (IFN-α/β) show strongly upregulated ISG15 expression

  • A549 or HepG2 cells treated with IFN-α are well-established positive controls

• Recombinant ISG15 protein: For antibody validation and as a size reference in Western blotting

• Transfected cells overexpressing tagged ISG15: Such as the HA-ISG15 system used in R&D Systems validation

Negative Controls:

• Non-stimulated cells: Cells without interferon treatment typically show minimal ISG15 expression

• ISG15 knockdown/knockout samples: To confirm antibody specificity

• Secondary antibody only: To identify any non-specific binding of the secondary antibody

Experimental Validation Controls:

• Multiple antibodies: Using antibodies targeting different epitopes helps confirm specificity

• Immunoprecipitation validation: As demonstrated in the R&D Systems data, comparing IP results from treated and untreated cells confirms specificity for ISGylated proteins

• Blocking peptide controls: Pre-incubation with immunizing peptide should abolish specific signals

These controls help distinguish between specific ISG15 signals and potential artifacts, ensuring reliable and reproducible results.

How do I optimize ISG15 antibody-based immunoprecipitation for studying protein ISGylation?

Optimizing immunoprecipitation of ISG15-conjugated proteins requires careful protocol design:

Sample Preparation:

• Stimulation: Treat cells with type I interferons to induce ISGylation

• Lysis conditions: Use denaturing lysis buffers containing SDS followed by dilution to reduce denaturation for IP, which helps maintain protein-protein interactions while preventing deISGylation by cellular proteases

Immunoprecipitation Protocol:

• Antibody selection: Choose antibodies validated for IP applications, such as R&D Systems A-830 or Proteintech 15981-1-AP

• Antibody amount: Use sufficient antibody (15-20 μg per 500 μg of lysate as demonstrated in R&D Systems protocol)

• Incubation conditions: Incubate overnight at 4°C with gentle rotation

• Bead selection: Protein G Sepharose is effective for rabbit polyclonal antibodies as demonstrated in the R&D Systems protocol

Detection and Validation:

• Western blotting: Blot with anti-ISG15 or antibodies against specific target proteins

• Complementary approaches: For tagged ISG15 systems, use anti-tag antibodies (e.g., anti-HA) for confirmation

• Sequential IP: For increased specificity, perform sequential IPs with different antibodies

The R&D Systems data demonstrates successful immunoprecipitation of ISGylated proteins from interferon-treated A549 cells, showing both free ISG15 and multiple higher molecular weight ISGylated proteins .

How can ISG15 antibodies be used to investigate the role of ISG15 in specific disease models?

ISG15 antibodies have proven valuable in studying disease associations through several advanced approaches:

Autoimmune Disease Research:

• In dermatomyositis (DM), ISG15 has been identified as a highly specific diagnostic biomarker with an AUC of 0.950 in ROC analysis

• Immunohistochemistry using ISG15 antibodies reveals universally positive ISG15 expression in DM patient skin samples, particularly in perivascular regions and lymphocytes, contrasting with negative expression in control tissues

• Analysis of ISG15 expression correlation with immune cell populations shows significant relationships with multiple immune cell types, suggesting regulatory roles in the disease microenvironment

Viral Infection Models:

• ISG15 antibodies can detect changes in ISGylation patterns during viral infection

• They help identify viral proteins targeted by ISG15, providing insights into viral evasion strategies

• Applications include studying ISG15's inhibition of HIV-1 and Ebola virus budding by disrupting ubiquitination of viral proteins

Cancer Research:

• ISG15 antibodies can evaluate ISG15 expression in various cancer types, as demonstrated by Proteintech's validation in human lung and pancreas cancer tissues

• They can help investigate the relationship between interferon signaling and tumor progression or response to immunotherapy

These applications demonstrate how ISG15 antibodies serve as powerful tools for understanding disease mechanisms beyond basic characterization of ISG15.

What are the latest methodologies for studying the dynamics of ISGylation and de-ISGylation?

Advanced methodologies for studying ISGylation dynamics include:

Real-time Monitoring Approaches:

• Fluorescent protein fusions with ISG15 for live-cell imaging

• FRET-based sensors to detect ISGylation-deISGylation events in real time

• Proximity ligation assays to visualize ISG15-target protein interactions in situ

Pulse-chase Experiments:

• Metabolic labeling with stable isotopes combined with IP and mass spectrometry to determine ISGylation/deISGylation rates

• Inducible expression systems to control ISG15 production and study temporal dynamics

Enzyme Activity Assays:

• In vitro ISGylation assays using recombinant E1 (UBE1L), E2 (UbcH8), and E3 (HERC5) enzymes

• DeISGylation assays using purified USP18/UBP43 to study removal kinetics

Proteomics Approaches:

• Tandem affinity purification of ISGylated proteins followed by mass spectrometry

• SILAC-based quantitative proteomics to compare ISGylated proteomes under different conditions

• Targeted proteomics focusing on known ISG15 targets like IFIT1, MX1/MxA, and PPM1B

These methods provide complementary data on the complex regulation of protein ISGylation and its biological significance in various cellular contexts.

How can I differentiate between free ISG15 and ISG15-conjugated proteins in experimental samples?

Differentiating between free ISG15 and ISG15-conjugated proteins requires specialized approaches:

Gel-based Separation Methods:

• Free ISG15 appears at 15-17 kDa on Western blots

• ISGylated proteins appear as higher molecular weight bands (typically >30 kDa)

• Gradient gels (4-20%) provide better resolution of the full range of ISGylated proteins

Antibody-based Strategies:

• Some antibodies are specifically validated for detecting ISGylated proteins in immunoprecipitation, such as R&D Systems A-830

• Using antibodies against specific target proteins followed by ISG15 detection can confirm ISGylation of particular proteins

Enzymatic Approaches:

• Treatment with deISGylating enzymes (USP18/UBP43) releases free ISG15 from conjugates

• Comparing samples before and after deISGylase treatment helps quantify conjugated vs. free ISG15

Expression Systems:

• Expressing tagged versions of ISG15 (HA-ISG15, FLAG-ISG15) allows differentiation using tag-specific antibodies

• The R&D Systems protocol demonstrates this approach using HA-tagged ISG15 in A549 cells

Controls and Validation:

• Include both interferon-treated samples (high ISGylation) and untreated controls (minimal ISGylation)

• Use cells expressing catalytically inactive ISG15 mutants that cannot form conjugates

These complementary approaches allow comprehensive analysis of ISG15 dynamics, distinguishing between its unconjugated form and its numerous protein conjugates.

What emerging research areas are utilizing ISG15 antibodies in novel ways?

ISG15 antibodies are being applied in several cutting-edge research areas:

• Single-cell analysis of ISG15 expression to understand cellular heterogeneity in interferon responses

• Spatial transcriptomics combined with ISG15 protein detection to map ISG15 activity in complex tissues

• Investigation of extracellular ISG15 functions beyond its intracellular conjugation roles

• Development of ISG15-targeted therapeutics for viral infections and autoimmune conditions

• Biomarker development for conditions like dermatomyositis where ISG15 shows diagnostic potential with high specificity (AUC 0.950)

These emerging applications highlight the continuing importance of high-quality ISG15 antibodies in advancing our understanding of this multifunctional protein in health and disease.

How do recent findings on ISG15 impact antibody selection and experimental design?

Recent discoveries about ISG15 biology have important implications for research approaches:

• Recognition of diverse functions: Beyond protein conjugation, ISG15 has non-conjugated functions that may require different detection strategies

• Species-specific differences: Significant functional differences exist between human and mouse ISG15, necessitating careful species consideration in experimental design

• Context-dependent expression: ISG15 expression patterns vary dramatically between tissue types and disease states, requiring optimization of detection protocols for each context

• Correlation with immune cell populations: The relationship between ISG15 and various immune cells, as demonstrated in dermatomyositis research, suggests the importance of multiparameter analysis approaches

• Drug development potential: The identification of drugs targeting ISG15, supported by molecular docking studies , opens new avenues for therapeutic research requiring specialized antibody applications