USP24 Antibody

What is USP24 and why is it significant in research?

USP24 (Ubiquitin Specific Peptidase 24) is a deubiquitinating enzyme (DUB) with a molecular weight of approximately 294 kDa that functions as a cysteine protease. This enzyme has garnered significant research interest due to its role in regulating cell survival through modulating the protein stability of several substrates including DDB2, MCL1, and TP53 . It plays a critical role in ferritinophagy, where ferritin is degraded in lysosomes to release free iron . Recent research has also identified USP24 as an ISG15 cross-reactive deubiquitinating enzyme that contributes to the regulation of interferon responses . The enzyme's involvement in cancer-related processes, particularly in lung cancer, makes it a valuable target for oncological research .

What applications are USP24 antibodies typically used for in research?

USP24 antibodies are versatile tools employed in multiple research applications:

These applications enable researchers to detect USP24 expression, localization, and interactions with other proteins in various experimental systems . The antibodies have been cited in numerous publications, primarily for Western blot, immunohistochemistry, immunofluorescence, and immunoprecipitation applications, as well as in knockdown/knockout validation studies .

How should USP24 antibody validation be performed when studying novel substrates?

Validating USP24 antibody specificity when investigating new substrates requires a multi-faceted approach:

• Antibody specificity validation: Begin with western blot analysis using positive controls (e.g., HepG2 cells, lung tissue) to confirm detection of bands at the expected molecular weight (280-294 kDa) . Include negative controls such as USP24 knockout or knockdown samples to verify specificity.

• Substrate-specific validation: For novel substrate studies, implement a stepwise validation process:

  • Perform co-immunoprecipitation (IP) with USP24 antibody followed by western blot for the putative substrate

  • Conduct reciprocal IP with the substrate's antibody and probe for USP24

  • Compare wild-type and USP24-depleted cells to examine changes in substrate ubiquitination levels

  • Conduct in vitro deubiquitination assays with recombinant USP24 and ubiquitinated substrate

• Functional validation: Examine the substrate protein stability in the presence and absence of USP24 through:

  • Cycloheximide chase assays to measure protein half-life

  • Proteasome inhibitor studies to determine degradation pathway

  • Site-directed mutagenesis of potential ubiquitination sites on the substrate

The E2F4 validation study serves as an excellent methodological template, where researchers demonstrated decreased E2F4 protein stability upon USP24 knockdown, followed by in vitro enzymatic assays showing decreased E2F4 ubiquitination in the presence of USP24 .

What are the critical considerations for immunohistochemical detection of USP24 in tissue samples?

For optimal immunohistochemical detection of USP24 in tissue samples, researchers should address several critical parameters:

• Antigen retrieval optimization: USP24 detection typically requires heat-induced epitope retrieval (HIER) with TE buffer at pH 9.0, although citrate buffer at pH 6.0 can serve as an alternative . Comparative testing of both methods is recommended for specific tissue types.

• Antibody dilution optimization: While a general range of 1:100-1:400 is recommended for IHC applications , titration experiments should be conducted for each tissue type, with serial dilutions tested to determine optimal signal-to-noise ratios.

• Specificity controls:

  • Positive control tissue (e.g., human lymphoma tissue)

  • Negative control (primary antibody omission)

  • Validation with a second antibody targeting a different epitope of USP24

  • USP24 knockdown or knockout tissue if available

• Signal amplification considerations: For tissues with low USP24 expression, polymerized reporter enzyme staining systems or tyramide signal amplification may be necessary.

• Counterstaining optimization: Adjust hematoxylin counterstaining time to ensure nuclear detail without obscuring specific USP24 staining.

• Multi-label approaches: For co-localization studies with substrate proteins like p53 or E2F4, sequential immunostaining protocols with appropriate controls for cross-reactivity are essential.

How does USP24's role in ISGylation contribute to interferon responses, and what experimental approaches can investigate this mechanism?

Recent research has identified USP24 as an ISG15 cross-reactive deubiquitinating enzyme that influences interferon responses through deISGylation of target proteins such as MOV10 . To investigate this mechanism:

• Identification of ISGylated substrates:

  • Activity-based probe profiling (ABPP) using propargylamide warhead probes to identify ISG15-reactive DUBs

  • RNAi-mediated depletion of USP24 combined with proteomics analyses:

    • Total proteome analysis

    • Enrichment with di-Gly antibody (GG-peptidome)

    • ISG15-specific antibody enrichment (ISG15 interactome)

    • Refined ISGylome analysis after USP24 deubiquitinase treatment

• Functional characterization:

  • Measure IFN-β secretion levels in USP24-depleted versus control cells

  • Assess ISGylation levels of specific targets (e.g., MOV10) in the presence/absence of USP24

  • Evaluate antiviral responses in cells with modified USP24 expression

• Mechanistic studies:

  • In vitro enzymatic assays to demonstrate direct deISGylation of targets by USP24

  • Structure-function analysis to identify domains responsible for ISG15 cross-reactivity

  • Site-directed mutagenesis of catalytic residues to confirm enzymatic activity

Evidence indicates that USP24 depletion leads to increased IFN-β secretion, suggesting a negative regulatory role in interferon responses . This positions USP24 as a potential therapeutic target in contexts where modulation of interferon responses would be beneficial, such as infectious diseases, cancer, autoimmunity, and neuroinflammation.

What are the approaches to resolve discrepancies in USP24 substrate identification across different cell types and experimental conditions?

Researchers frequently encounter discrepancies in USP24 substrate identification across different experimental systems. To systematically address these variations:

• Comprehensive multi-omics approach:

  • Compare UPS24 interactomes across different cell types using IP-MS

  • Conduct parallel ubiquitinome analyses in multiple cell types after USP24 depletion

  • Integrate transcriptomics data to account for cell-type-specific expression differences

• Context-dependent activation studies:

  • Examine USP24 activity under various cellular stresses (oxidative stress, DNA damage, ER stress)

  • Analyze post-translational modifications of USP24 that might alter substrate specificity

  • Investigate USP24 subcellular localization changes that could affect substrate accessibility

• Methodological standardization:

  • Implement consistent USP24 depletion methods (siRNA, shRNA, CRISPR) with validation

  • Standardize detection methods for ubiquitinated proteins

  • Document key experimental variables:

    • Cell confluency

    • Passage number

    • Culture conditions

    • Harvest and lysis methods

• Resolution strategies for conflicting data:

  • Direct comparison studies with standardized protocols

  • Dose-response experiments with varying USP24 expression levels

  • Kinetic studies to capture temporal dynamics of deubiquitination

For example, USP24's role in E2F4 deubiquitination in lung cancer cells might differ from its function in other contexts, such as its involvement in ISGylation and interferon responses . By implementing these approaches, researchers can disambiguate cell-type-specific functions from universal USP24 mechanisms.

What strategies can minimize non-specific binding when using USP24 antibodies in complex tissue samples?

When working with complex tissue samples, non-specific binding can significantly compromise USP24 detection specificity. Implement these strategies to improve signal-to-noise ratio:

• Blocking optimization:

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

  • Test extended blocking times (2-16 hours)

  • Evaluate concentration-dependent effects of blocking reagents

  • Consider dual-blocking approaches (protein block followed by peroxidase/phosphatase block)

• Antibody preparation techniques:

  • Pre-adsorption with tissue homogenates from USP24-knockout models

  • Affinity purification against the immunizing peptide

  • Testing multiple antibody clones targeting different USP24 epitopes

  • Optimization of antibody incubation conditions (temperature, time, buffer composition)

• Sample-specific considerations:

  • Optimize fixation protocols (duration, fixative composition)

  • Implement tissue-specific antigen retrieval modifications

  • Adjust permeabilization conditions for different tissue densities

  • Use Sudan Black B to reduce autofluorescence in immunofluorescence applications

• Advanced detection strategies:

  • Implement tyramide signal amplification for weak signals

  • Consider proximity ligation assays for improved specificity

  • Use spectral imaging and unmixing for autofluorescent tissues

  • Employ super-resolution microscopy techniques for specific applications

The recommended antibody dilution ranges (1:100-1:400 for IHC, 1:50-1:500 for IF/ICC) should serve as starting points for optimization experiments specific to each tissue type and application.

How can USP24 antibodies be optimized for use in live cell imaging studies?

Adapting USP24 antibodies for live cell imaging presents significant challenges due to the need to maintain cell viability while achieving specific labeling. Consider these optimization strategies:

• Antibody fragment generation:

  • Produce Fab or scFv fragments from full IgG USP24 antibodies

  • Verify retained specificity through fixed-cell immunostaining

  • Assess membrane permeability of fragments in live cells

  • Optimize fragment concentration to balance signal and toxicity

• Fluorophore selection and conjugation:

  • Select bright, photostable fluorophores with minimal phototoxicity

  • Optimize dye-to-antibody ratios to prevent aggregation

  • Consider pH-sensitive fluorophores for endosomal tracking

  • Test multiple conjugation chemistries to preserve epitope recognition

• Delivery optimization:

  • Evaluate protein transfection reagents for antibody delivery

  • Assess electroporation parameters for different cell types

  • Test cell-penetrating peptide conjugation approaches

  • Optimize microinjection techniques for sensitive cell types

• Validation and controls:

  • Compare live-cell staining patterns with fixed-cell immunostaining

  • Use USP24-GFP fusion proteins as positive controls

  • Implement CRISPR-mediated tagging of endogenous USP24

  • Perform competition experiments with unlabeled antibodies

• Imaging parameters:

  • Determine optimal acquisition settings to minimize phototoxicity

  • Implement oxygen scavenging systems for extended imaging

  • Utilize deconvolution and computational approaches to enhance signal

  • Consider lattice light-sheet microscopy for extended 3D imaging

While conventional USP24 antibodies are primarily validated for fixed-cell applications , these optimization strategies may enable adaptation for specific live-cell imaging requirements.

How can USP24 antibodies be leveraged in studying the intersection of deubiquitination and cancer immunotherapy resistance?

Emerging research suggests potential connections between deubiquitination processes and immunotherapy responses. USP24 antibodies can be instrumental in exploring this intersection:

• Profiling USP24 expression in responders vs. non-responders:

  • Implement multiplex immunohistochemistry to co-localize USP24 with immune checkpoint molecules (PD-L1, CTLA-4) in patient biopsies

  • Correlate USP24 expression patterns with treatment outcomes using tissue microarrays

  • Develop quantitative image analysis workflows for objective assessment

• Mechanistic investigations:

  • Study USP24's impact on stability of immune checkpoint molecules using cycloheximide chase assays

  • Investigate USP24-mediated deubiquitination of key immune signaling components

  • Examine USP24's role in regulating interferon responses in tumor microenvironments, building on its established function in IFN-β regulation

• Therapeutic targeting assessment:

  • Use USP24 antibodies to monitor protein levels during treatment with DUB inhibitors

  • Develop proximity-based assays to screen for compounds disrupting USP24-substrate interactions

  • Establish correlation between USP24 inhibition and immunotherapy sensitization

• Translational applications:

  • Develop companion diagnostic approaches using validated USP24 IHC protocols

  • Create standardized reporting systems for USP24 expression in tumor samples

  • Establish predictive biomarker potential through retrospective and prospective studies

The previously established connections between USP24 and cancer-related proteins like Bax and p300 , combined with its newly discovered role in ISGylation and interferon responses , provide a strong foundation for investigating its impact on immunotherapy outcomes.

What approaches can effectively characterize post-translational modifications of USP24 and their impact on its enzyme activity?

Post-translational modifications (PTMs) can significantly modulate USP24's enzymatic activity, substrate specificity, and cellular localization. To comprehensively characterize these modifications:

• Global PTM profiling strategies:

  • Immunoprecipitate endogenous USP24 using validated antibodies followed by mass spectrometry

  • Compare PTM profiles across different cell types and treatment conditions

  • Implement complementary enrichment strategies for specific PTMs (phosphorylation, acetylation, SUMOylation)

  • Consider top-down proteomics approaches to preserve modification relationships

• Functional impact assessment:

  • Generate site-specific mutants of identified PTM sites

  • Conduct in vitro deubiquitination assays with modified vs. unmodified USP24

  • Analyze substrate binding affinity changes using surface plasmon resonance

  • Perform structural studies to determine how PTMs affect catalytic domain conformation

• Regulatory enzyme identification:

  • Conduct candidate-based screens of kinases, phosphatases, acetyltransferases

  • Perform proximity labeling approaches (BioID, APEX) to identify proteins in close proximity to USP24

  • Use pharmacological modulators of PTM-placing enzymes to confirm relationships

  • Develop FRET-based sensors to monitor USP24 conformational changes upon modification

• Physiological context determination:

  • Examine PTM patterns during cell cycle progression

  • Analyze modifications in response to cellular stresses (DNA damage, oxidative stress)

  • Investigate PTM changes during viral infection, connecting to USP24's role in interferon responses

  • Compare modification patterns in normal vs. cancer cells, particularly in lung cancer contexts

Understanding USP24 PTMs may provide insights into additional regulatory mechanisms beyond substrate availability and protein expression, potentially identifying novel therapeutic opportunities for conditions where USP24 activity contributes to pathology.

How can researchers address weak or inconsistent Western blot signals when using USP24 antibodies?

Given USP24's high molecular weight (280-294 kDa) , researchers often encounter challenges with Western blot detection. Implement these troubleshooting strategies for improved results:

Additional optimization considerations include selecting the appropriate gel percentage (4-6% for resolving high MW proteins), extending SDS-PAGE running time, and implementing gradient gels to improve separation in the high molecular weight range.

What strategies can improve USP24 detection in cells with low endogenous expression?

Detecting USP24 in cells with low endogenous expression presents significant technical challenges. Consider these approaches to enhance detection sensitivity:

• Sample preparation optimization:

  • Implement subcellular fractionation to concentrate USP24 from relevant compartments

  • Use immunoprecipitation to enrich USP24 prior to analysis

  • Test different lysis buffers optimized for nuclear protein extraction

  • Consider KRAS pathway activation to potentially upregulate USP24 expression

• Signal amplification techniques:

  • For Western blotting:

    • Implement enhanced chemiluminescence substrates with extended sensitivity

    • Consider biotin-streptavidin amplification systems

    • Use secondary antibody polymer conjugates for increased signal

    • Extend exposure times with cooled CCD camera systems

  • For immunofluorescence:

    • Implement tyramide signal amplification (TSA)

    • Use quantum dot conjugated secondary antibodies

    • Consider amplification systems like CARD or SABER

    • Optimize image acquisition with sensitive EMCCD cameras

• Alternative detection approaches:

  • Implement proximity ligation assays (PLA) to detect USP24 interactions

  • Consider RNAscope® for mRNA detection if protein levels are below detection

  • Use CRISPR-mediated endogenous tagging with bright fluorescent proteins

  • Implement mass spectrometry-based targeted proteomics approaches

• Positive controls and validation:

  • Include cells with known USP24 expression (HepG2, lung tissue)

  • Implement transient overexpression controls

  • Use IFN-β stimulation which may increase USP24 detection through pathway activation

  • Verify specificity with siRNA knockdown controls

By implementing these strategies, researchers can overcome detection limitations and successfully study USP24 biology even in systems with naturally low expression levels.

What are the emerging applications of USP24 antibodies in translational research?

USP24 antibodies are increasingly being employed in translational research contexts that bridge basic science and clinical applications:

• Biomarker development:

  • Standardized immunohistochemistry protocols for patient stratification

  • Correlation studies linking USP24 expression patterns with disease progression

  • Multiplexed imaging approaches combining USP24 with other cancer markers

  • Liquid biopsy applications for circulating tumor cell USP24 detection

• Therapeutic development:

  • Target engagement studies for USP24 inhibitor development

  • Pharmacodynamic marker applications in clinical trials

  • Resistance mechanism investigations in targeted therapies

  • Patient selection strategies for precision medicine approaches

• Disease mechanism elucidation:

  • USP24's role in lung cancer progression through E2F4 deubiquitination

  • Connections between USP24 and interferon responses in inflammatory diseases

  • Investigation of USP24 in neurodegeneration and neuroinflammation

  • Exploring USP24's function in cellular iron homeostasis through ferritinophagy regulation

• Technical innovations:

  • Development of conformation-specific antibodies to detect active vs. inactive USP24

  • Implementation of intrabodies for real-time monitoring of USP24 activity

  • Creation of activity-based probes incorporating USP24 antibody fragments

  • Nanoparticle conjugation strategies for targeted delivery in therapeutic applications

The dual role of USP24 in deubiquitination and deISGylation positions it at the intersection of multiple cellular pathways with significant therapeutic potential, making USP24 antibodies invaluable tools for translational research.

How should researchers integrate USP24 antibody-based techniques with emerging technologies like spatial transcriptomics and single-cell proteomics?

As molecular analysis technologies evolve, integration of USP24 antibody-based techniques with advanced platforms presents both opportunities and methodological challenges:

• Integration with spatial transcriptomics:

  • Develop sequential immunofluorescence and in situ hybridization protocols

  • Implement computational approaches to correlate USP24 protein localization with spatial gene expression patterns

  • Establish multiplexed protein-RNA co-detection methods for USP24 and its substrates

  • Study spatial relationships between USP24 and substrate mRNAs in different microenvironments

• Single-cell proteomics applications:

  • Adapt USP24 antibodies for CyTOF/mass cytometry applications

  • Develop protocols for antibody-based single-cell Western blotting

  • Implement microfluidic platforms for single-cell proteomic analysis with USP24 detection

  • Create optimized workflows for CITE-seq incorporating USP24 antibodies

• Multi-omics data integration:

  • Establish computational frameworks to correlate USP24 protein levels with transcriptome, ubiquitinome, and ISGylome data

  • Develop visualization tools for multi-parameter USP24 pathway analysis

  • Implement machine learning approaches to identify patterns in USP24-related multi-omics datasets

  • Create integrated databases of USP24 interactions across multiple data types

• Technical considerations:

  • Optimize fixation and permeabilization protocols compatible with both protein and RNA detection

  • Develop antibody validation strategies specific to multi-omics applications

  • Establish quality control metrics for USP24 detection in complex workflows

  • Create benchmarking standards for cross-platform comparisons