USP50 Antibody

What is USP50 and what cellular functions does it regulate?

USP50 (ubiquitin specific peptidase 50) belongs to the peptidase C19 family and functions as a deubiquitinating enzyme. Research has established that USP50 plays critical roles in multiple cellular processes, particularly in cell cycle regulation and DNA repair mechanisms through its interaction with the Wee1 protein in transformed human osteosarcoma and epithelial cell lines. Additionally, USP50 contributes to inflammasomal function by targeting specific adaptor proteins . The protein has a calculated molecular weight of 39 kDa, though it typically appears at approximately 40 kDa in experimental contexts, potentially due to post-translational modifications . This understanding of USP50's functional roles provides the foundation for experimental design when investigating its activities in various cellular contexts.

What applications are USP50 antibodies validated for in research settings?

USP50 antibodies have been validated for multiple experimental applications in research settings, with different antibody clones showing slightly different application profiles. The predominant validated applications include:

When selecting an antibody for specific research applications, researchers should consider the documented validation data for each application as well as the species reactivity required for their experimental system . These validation data ensure that experimental results accurately reflect USP50 presence and function.

What tissue and cell types express detectable levels of USP50 for antibody-based detection?

Based on validation studies, USP50 has been reliably detected in several tissue and cell types, making them suitable positive controls for antibody-based detection methods:

These validated sample types provide researchers with reliable positive controls for establishing experimental protocols. When transitioning to different tissue or cell types, appropriate validation steps should be implemented to confirm antibody performance in the new experimental context . Researchers should note that expression levels may vary across different tissues, necessitating optimization of detection protocols.

What are the optimal dilution ratios for different USP50 antibody applications?

The optimal dilution ratios for USP50 antibodies vary by both application type and specific antibody clone. Based on manufacturer recommendations and published research, the following dilution ranges have been established:

It is critical to note that these ranges represent starting points for optimization. The manufacturer guidance emphasizes that "this reagent should be titrated in each testing system to obtain optimal results" and that optimal dilutions may be "sample-dependent" . Best practice involves conducting a dilution series during protocol establishment to determine the optimal signal-to-noise ratio for specific experimental conditions, which may vary based on tissue type, fixation method, or detection system.

What antigen retrieval methods are recommended for USP50 immunohistochemistry?

For optimal antigen retrieval in USP50 immunohistochemistry applications, manufacturers recommend a primary protocol using TE buffer at pH 9.0. This alkaline pH retrieval method has been validated across multiple tissue types including human hepatocirrhosis tissue and mouse testis tissue. As an alternative when this primary method yields suboptimal results, citrate buffer at pH 6.0 has been suggested as a secondary option .

The effectiveness of antigen retrieval can vary significantly based on tissue fixation conditions and processing methods. For formalin-fixed paraffin-embedded (FFPE) human skeletal muscle samples specifically, successful immunohistochemistry detection has been documented following appropriate antigen retrieval, peroxidase conjugation of secondary antibodies, and DAB staining . Researchers should systematically evaluate both recommended antigen retrieval methods when establishing protocols for previously untested tissue types.

How should USP50 antibodies be stored to maintain optimal activity?

Proper storage conditions are essential for maintaining antibody activity and experimental reproducibility. For USP50 antibodies, manufacturers recommend:

The storage buffer for these antibodies typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. This formulation helps maintain antibody stability during freeze-thaw cycles. Importantly, manufacturers note that aliquoting is unnecessary for -20°C storage, which differs from common practice for many other antibodies . For the 20μl size formulations, it's worth noting that they contain 0.1% BSA, which may impact certain applications requiring BSA-free conditions.

How can specificity of USP50 antibodies be validated in experimental systems?

Validating antibody specificity is crucial for ensuring experimental rigor. For USP50 antibodies, a multi-faceted validation approach is recommended:

• Knockout/Knockdown Controls: Several publications have documented the use of USP50 knockdown/knockout systems for antibody validation. This approach represents the gold standard for specificity confirmation, as the disappearance or significant reduction of signal in KD/KO samples provides strong evidence of antibody specificity .

• Molecular Weight Verification: USP50 has a calculated molecular weight of 39 kDa but is typically observed at approximately 40 kDa on Western blots. Researchers should verify that their antibody detects a protein of this expected size .

• Positive Control Tissues: Using validated positive control tissues such as mouse testis, mouse brain tissue, or Y79 cell lines provides an additional layer of validation .

• Blocking Peptides: For peptide-generated antibodies like 20374-1-AP, competition assays using the immunizing peptide can provide additional evidence of specificity.

The implementation of multiple validation approaches significantly strengthens confidence in experimental results and should be considered essential rather than optional for rigorous research applications.

What computational approaches exist for predicting and designing antibody specificity profiles for USP50?

Recent advances in computational biology have enabled more sophisticated approaches to antibody specificity prediction and design. For researchers interested in customizing antibody specificity profiles for USP50 detection, computational models based on phage display experiments have demonstrated promising results.

These computational approaches focus on identifying distinct binding modes associated with particular epitopes, allowing for the design of antibodies with either highly specific binding to singular targets or cross-reactivity across multiple related targets. The methodology involves:

• Analyzing high-throughput sequencing data from phage display experiments

• Identifying binding modes associated with specific epitopes

• Building energy functions (E) that describe binding preferences

• Optimizing sequences to either minimize energy functions (for cross-specificity) or to minimize for desired targets while maximizing for undesired targets (for high specificity)

This approach has proven particularly valuable when working with chemically similar epitopes that traditional experimental methods struggle to differentiate. Researchers can leverage these computational models to design antibodies with customized specificity profiles beyond what is achievable through traditional selection methods alone, potentially enabling more precise targeting of USP50 variants or isoforms .

How can researchers distinguish between USP50 isoforms in experimental systems?

USP50 has been documented to have at least two isoforms, which presents challenges for experimental design and data interpretation. To effectively distinguish between these isoforms:

• Antibody Selection: Choose antibodies generated against epitopes that differ between isoforms. For example, the 102-11015 antibody specifically targets the central region (amino acids 141-169) of human USP50, which may be differentially present in certain isoforms .

• Multiple Detection Methods: Implement complementary techniques such as Western blotting (for size discrimination) alongside immunofluorescence (for localization patterns), as isoforms may exhibit different subcellular distributions.

• RNA Analysis: Complement protein-level detection with isoform-specific PCR primers to correlate transcript and protein expression patterns.

• Mass Spectrometry: For definitive identification, consider using immunoprecipitation followed by mass spectrometry analysis to identify isoform-specific peptides.

Careful experimental design that incorporates multiple detection modalities is essential when investigating USP50 isoforms to avoid misinterpretation of results that might arise from isoform-specific antibody reactivity patterns.

What special considerations apply when detecting USP50 in reproductive tissues?

USP50 detection in reproductive tissues, particularly testis, requires special methodological considerations:

• Fixation Protocol Optimization: Testicular tissue contains multiple cell types at various stages of spermatogenesis, each potentially expressing USP50 at different levels. Fixation duration and conditions significantly impact epitope preservation and should be carefully optimized.

• Background Reduction: Testicular tissue often exhibits higher background staining in immunohistochemistry applications. For USP50 antibodies, optimization of blocking protocols is particularly important, with testing of various blocking agents (BSA, normal serum, commercial blockers) recommended.

• Stage-Specific Analysis: When analyzing USP50 expression in testis, researchers should consider stage-specific expression patterns within seminiferous tubules, which requires careful anatomical identification and documentation.

Both human and mouse testis tissues have been validated as positive controls for USP50 detection using antibodies 24817-1-AP and 20374-1-AP, making these tissues reliable positive controls for protocol establishment . The consistent detection of USP50 in testicular tissue suggests important functional roles in reproductive biology that warrant dedicated investigation.

What approaches are recommended for detecting USP50 in neurological tissues?

Detection of USP50 in neurological tissues presents unique challenges that require methodological adaptations:

• Signal Amplification: Mouse brain tissue has been validated for USP50 detection by Western blot using antibody 20374-1-AP, but may require enhanced signal amplification methods for optimal visualization due to potentially lower expression levels compared to testicular tissue .

• Regional Analysis: Brain tissue exhibits significant regional heterogeneity, necessitating careful anatomical documentation when reporting USP50 expression patterns. researchers should specify brain regions examined rather than referring generally to "brain tissue."

• Cell Type Identification: Neurological tissues contain multiple cell types (neurons, astrocytes, microglia, oligodendrocytes), requiring co-staining with cell-type specific markers to determine the cellular source of USP50 expression.

• Antigen Retrieval Optimization: For fixed brain tissue sections, antigen retrieval methods may require additional optimization beyond the standard recommendations, potentially including extended incubation times or alternative buffer compositions.

Considering USP50's established roles in DNA repair and cell cycle regulation, its expression in post-mitotic neurons versus proliferative cells within the nervous system may provide insights into tissue-specific functions that extend beyond its previously documented roles.

How can USP50 antibodies be integrated with functional deubiquitination assays?

As a member of the ubiquitin-specific peptidase family, USP50's primary biochemical function involves deubiquitination activity. Integrating antibody-based detection with functional assays requires methodological considerations:

• Activity-State Specific Detection: Consider whether the antibody recognizes active, inactive, or both states of USP50. Antibodies generated against different regions may exhibit differential detection based on conformational states.

• Immunoprecipitation-Activity Assays: USP50 antibodies can be used to immunoprecipitate the enzyme, followed by in vitro deubiquitination assays using synthetic substrates or purified ubiquitinated proteins.

• Correlation Analysis: When analyzing USP50 expression levels via antibody-based methods, parallel assessment of deubiquitinating activity in the same samples can establish correlations between protein abundance and enzymatic function.

• Inhibitor Studies: USP50 antibodies can be used to monitor protein levels in response to treatment with deubiquitinase inhibitors, providing insights into potential compensatory mechanisms or protein stability alterations.

The integration of quantitative antibody-based detection methods with functional enzymatic assays provides a more comprehensive understanding of USP50 biology than either approach alone, particularly when investigating potential disruptions in ubiquitin homeostasis.

What approaches are recommended for studying USP50 interactions with Wee1 and cell cycle regulation?

Given USP50's documented interaction with Wee1 and its role in cell cycle regulation, several methodological approaches are recommended for investigating these relationships:

• Co-Immunoprecipitation: USP50 antibodies can be used in co-immunoprecipitation studies to pull down USP50 and associated proteins, with subsequent detection of Wee1 to confirm their interaction under various experimental conditions.

• Proximity Ligation Assays: For visualizing USP50-Wee1 interactions in situ, proximity ligation assays using USP50 and Wee1 antibodies can provide spatial information about their interaction with subcellular resolution.

• Cell Cycle Synchronization: When studying USP50's role in cell cycle regulation, combining cell synchronization protocols with time-course antibody-based detection can reveal cell cycle-dependent changes in USP50 expression, localization, or interaction patterns.

• Phosphorylation Status: Since Wee1 is regulated by phosphorylation, researchers should consider using phospho-specific antibodies alongside USP50 detection to correlate USP50 activity with Wee1 phosphorylation status.

These approaches can be particularly informative in transformed human osteosarcoma and epithelial cell lines, where USP50's interaction with Wee1 has been previously established . The combination of biochemical and imaging-based techniques provides complementary insights into the functional significance of USP50 in cell cycle regulatory networks.

How can USP50 antibodies be utilized to investigate its role in inflammasome function?

Recent research has identified USP50's involvement in inflammasome function through its interaction with adaptor proteins . To investigate this emerging functional role:

• Co-localization Studies: Implement dual immunofluorescence using USP50 antibodies alongside antibodies against key inflammasome components (NLRP3, ASC, caspase-1) to assess spatial relationships during inflammasome activation.

• Temporal Dynamics: Design time-course experiments using USP50 antibodies to monitor expression and localization changes during inflammasome activation induced by canonical stimuli (ATP, nigericin, etc.).

• Inflammasome Fractionation: Apply USP50 antibodies in Western blot analysis of biochemically fractionated inflammasome complexes to determine USP50's association with different subcomplexes during assembly and activation.

• Ubiquitination Analysis: Combine USP50 detection with ubiquitin-specific antibodies to assess the ubiquitination status of inflammasome components in the presence or absence of functional USP50.

These methodological approaches can help elucidate the specific mechanisms by which USP50 influences inflammasome regulation, potentially identifying novel therapeutic targets for inflammatory diseases where inflammasome dysregulation plays a pathogenic role.

What are the recommended approaches for investigating USP50 in the context of DNA damage response pathways?

Given USP50's established role in DNA repair processes, several methodological approaches are recommended for investigating its functions in DNA damage response:

• Damage-Induced Dynamics: Monitor USP50 localization changes following induction of DNA damage using immunofluorescence with validated USP50 antibodies (e.g., 20374-1-AP for IF/ICC applications) .

• Chromatin Association: Implement chromatin fractionation protocols followed by Western blotting with USP50 antibodies to determine damage-induced chromatin association patterns.

• Repair Foci Co-localization: Perform co-immunofluorescence studies with USP50 antibodies and antibodies against DNA repair factors (γH2AX, 53BP1, RAD51, etc.) to assess recruitment to DNA damage sites.

• Repair Pathway Specificity: Design experiments using pathway-specific DNA damaging agents (e.g., camptothecin for replication-associated breaks, neocarzinostatin for DSBs) to determine if USP50 exhibits damage-type specific responses.

Implementation of these approaches can provide insights into USP50's specific contributions to DNA repair pathways, potentially revealing novel regulatory mechanisms in genome maintenance systems with implications for cancer biology and therapeutic resistance.