USP18 Antibody, Biotin conjugated

What is USP18 and what biological functions does it have?

USP18 functions as a multifunctional cysteine protease primarily responsible for deconjugating the interferon-inducible ubiquitin-like modifier ISG15 from protein substrates. It plays a critical role in regulating immune responses to viral infections and other inflammatory stimuli through two key mechanisms. First, USP18 acts enzymatically by deISGylating target proteins, removing the ISG15 modification that occurs during immune activation. Second, it functions non-enzymatically by inhibiting the interferon signaling pathway, providing negative feedback regulation of immune responses .

Recent research has revealed that USP18 can also function as a scaffold protein to facilitate the re-localization of TRIM31 and enhance the interaction between TRIM31 and MAVS in mitochondria, positively regulating innate antiviral immunity . Additionally, mutations in the USP18 gene have been linked to autoimmune disorders such as systemic lupus erythematosus (SLE) and type 1 diabetes, as well as increased susceptibility to viral infections .

What applications are recommended for USP18 Antibody, Biotin conjugated?

The USP18 Antibody, Biotin conjugated (Product code: CSB-PA891559LD01HU) is specifically recommended for ELISA applications as indicated in the product documentation . This biotin conjugation provides enhanced sensitivity in detection systems utilizing streptavidin-based visualization methods. While this specific conjugate is optimized for ELISA, the parent antibody (non-conjugated form, CSB-PA891559LA01HU) can be used across multiple applications including Western Blot (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF) with the following recommended dilution ranges:

The biotin conjugation makes this antibody particularly valuable for multi-step detection protocols where signal amplification is desired .

What are the key considerations for validating USP18 antibody specificity?

Validating the specificity of USP18 antibodies requires careful experimental design to ensure accurate results. The USP18 antibody used in research was derived from rabbits immunized with the recombinant human USP18 protein (18-118aa region) and has been demonstrated to recognize both human and mouse USP18 proteins . When validating this antibody, researchers should:

First, include appropriate positive controls using cell lines known to express USP18, particularly those treated with type I interferons to upregulate USP18 expression. Second, negative controls should include cells where USP18 has been knocked out or knocked down using CRISPR or siRNA technologies. Third, cross-reactivity testing against other related USPs (particularly USP5 and USP14) is essential since these show some structural similarity to USP18 . Finally, comparison of results across multiple detection methods (ELISA, WB, IHC) can provide additional validation of specificity.

It's worth noting that the parent antibody reaches up to 95% purity after protein G purification, contributing to its specificity profile .

How should samples be prepared for optimal USP18 detection using the biotin-conjugated antibody?

For optimal detection of USP18 using the biotin-conjugated antibody in ELISA applications, sample preparation should account for the biological properties of USP18 and its cellular distribution. Since USP18 shows differential localization between mitochondria and endoplasmic reticulum depending on cellular stimulation conditions , careful subcellular fractionation may be necessary for certain research questions.

For cell lysate preparation, samples should be collected in a buffer containing protease inhibitors to prevent degradation of USP18. For detection of endogenous USP18, stimulation with type I interferons is often necessary as USP18 is an interferon-stimulated gene (ISG). The protein level of USP18 has been observed to increase specifically in mitochondria rather than the endoplasmic reticulum following RNA virus infection , which suggests that mitochondrial enrichment may be beneficial for certain experimental designs.

When analyzing clinical samples, consideration should be given to USP18's differential expression across cancer types, as lower USP18 expression correlates with better survival in certain cancers .

What protein interactions should be considered when studying USP18?

When studying USP18 using antibody-based detection methods, researchers should consider several key protein interactions that may influence experimental results. USP18 has been shown to interact with:

First, ISG15 and ISGylated proteins represent the primary substrates for USP18's enzymatic activity. The biotin-conjugated antibody can be particularly useful for co-immunoprecipitation studies examining these interactions . Second, components of the interferon signaling pathway, including STATs and IFN receptors, interact with USP18 in its role as a negative regulator. Third, MAVS (Mitochondrial antiviral-signaling protein) interaction is critical, as USP18 has been shown to enhance MAVS ubiquitination rather than reduce it, contrary to its typical deubiquitinating function .

Additionally, USP18 has been found to recruit another DUB, USP20, to deconjugate K33- and K48-linked polyubiquitin chains from STING, demonstrating the complexity of its protein interaction network . Understanding these interactions is essential for properly interpreting results obtained with the USP18 antibody.

How can activity-based probes be used alongside antibodies to study USP18 function?

Activity-based probes (ABPs) provide complementary approaches to antibody-based detection by specifically measuring enzymatic activity rather than just protein presence. For USP18 research, specialized ABPs have been designed that incorporate unnatural amino acids into the C-terminal tail of ISG15, enabling selective detection of USP18 activity over other ISG15 cross-reactive deubiquitinases such as USP5 and USP14 .

To implement a comprehensive study of USP18, researchers should consider a dual approach. The biotin-conjugated USP18 antibody can be used for protein quantification and localization studies, while ISG15-based ABPs can measure enzymatic activity. This combination allows discrimination between changes in USP18 protein levels versus changes in its catalytic activity. Importantly, recent research has developed a chemoproteomics screening platform that combines USP18 ABPs with ubiquitin-based DUB ABPs to identify and assess inhibitors of DUBs including USP18 .

When employing this dual approach, it's important to note that some USP18 functions are enzymatic-independent, acting as a scaffold protein, which would be detected by antibodies but not by activity-based probes .

What methodological approaches can distinguish between USP18's enzymatic and non-enzymatic functions?

Distinguishing between USP18's enzymatic deISGylase activity and its non-enzymatic scaffolding functions requires carefully designed experimental protocols. A multi-faceted approach is recommended:

First, comparative studies using wild-type USP18 and catalytically inactive mutants (e.g., USP18 C61A) can help differentiate between these functions. Previous research has shown that the antiviral ability of Usp18 C61A cells was comparable to wild-type cells against herpes simplex virus 1 infection, suggesting that the enzymatic activity of USP18 is dispensable for some antiviral responses .

Second, subcellular localization studies using fractionation followed by immunoblotting with the USP18 antibody can help identify compartment-specific functions, as USP18 protein levels have been observed to increase specifically in mitochondria rather than the endoplasmic reticulum following RNA virus infection .

Third, protein interaction studies using proximity ligation assays or co-immunoprecipitation with the USP18 antibody can identify binding partners involved in either enzymatic or scaffolding functions. Fourth, using the biotin-conjugated antibody in combination with streptavidin pull-down can help identify novel USP18 complexes that mediate its non-enzymatic functions.

How can USP18 antibodies be used to investigate its role in cancer immunotherapy?

USP18 has emerged as a potential target for cancer immunotherapy, with recent research showing that depletion of USP18 selectively induces cancer cell immunogenic cell death (ICD) . To investigate this therapeutic potential, several methodological approaches using USP18 antibodies are recommended:

First, immunohistochemical profiling of patient tumor samples using the USP18 antibody can help establish correlations between USP18 expression levels and clinical outcomes, building on findings that lower USP18 expression correlates with better survival across selected human cancer types .

Second, mechanistic studies should focus on nuclear USP18, as research has revealed that nuclear USP18 controls the enhancer landscape of cancer cells and diminishes STAT2-mediated transcription complex binding to IFN-responsive elements . This can be accomplished through subcellular fractionation followed by immunoblotting or chromatin immunoprecipitation using the USP18 antibody.

Third, single-cell analyses incorporating USP18 antibody-based detection can help investigate the finding that inhibition of USP18 specifically targets cancer stem cells essential for relapse . Fourth, combination therapy studies should examine USP18 in the context of STING agonist-based immunotherapy, as the survival benefit of USP18 depletion was significantly enhanced with the addition of DMXAA (a STING agonist) .

What controls are essential when studying cross-reactivity between USP18 and other DUBs?

When investigating potential cross-reactivity between USP18 and other deubiquitinases (DUBs), several critical controls must be incorporated:

First, competition assays between ISG15 and ubiquitin substrates should be conducted to assess specificity, as several DUBs including USP2, USP5, USP13, USP14, and USP21 have been shown to react covalently with ISG15-based probes due to structural similarities .

Second, parallel testing with multiple DUB antibodies is essential, particularly for USP5, USP14, and USP16, which have been identified as ISG15 cross-reactive DUBs in unbiased activity-based probe profiling studies . Third, interferon stimulation controls are crucial, as USP18 was the only hISG15CTD-PA-reacting protease significantly enriched in IFN-stimulated cells compared to non-stimulated cells .

Fourth, substrate specificity controls using known targets of both USP18 and other DUBs can help differentiate their biological functions. For example, MDH1 can be ISGylated by the UBE1L-UBCH8-HERC5 cascade and is subject to USP16-dependent cleavage .

The biotin-conjugated USP18 antibody can be particularly valuable in these studies for pull-down experiments followed by activity assays to isolate USP18 complexes and test their specificity against various substrates.

How can USP18 antibodies be integrated into studies of interferon signaling regulation?

Integrating USP18 antibodies into interferon signaling studies requires careful experimental design to capture USP18's complex regulatory functions. A comprehensive approach should include:

First, time-course studies using the biotin-conjugated USP18 antibody in ELISA format can track the dynamic expression of USP18 following interferon stimulation, providing insights into the temporal regulation of the interferon response .

Second, chromatin immunoprecipitation (ChIP) assays using USP18 antibodies can investigate its nuclear role in diminishing binding of IFN-regulated transcription factors to their corresponding DNA motifs, as recent research has uncovered that nuclear USP18 controls both typical ISGs and non-canonical ISGs important for cancer cell pyroptosis .

Third, protein complex analysis through co-immunoprecipitation with USP18 antibodies can identify interactions with STAT2 and NF-κB, as USP18 has been shown to cooperate with NF-κB to regulate the binding of IFN-regulated transcription factors .

Fourth, comparative studies between wild-type and USP18-depleted cells using single-cell RNA sequencing can help identify USP18-dependent changes in the interferon-stimulated gene landscape, building on findings that USP18 suppression enhances expression of interferon-stimulated genes like ISG15 across all cell clusters .