UCHL1 Antibody

What is UCHL1 and why is it important in neuroscience research?

UCHL1 (also known as PGP 9.5, PGP9.5, Protein Gene Product 9.5, HEL-117, or HEL-S-53) is a 24.8 kilodalton protein that plays crucial roles in regulating cellular free ubiquitin levels, redox state, and the degradation of select proteins . UCHL1 is particularly important in neuroscience research because:

• It constitutes 1-2% of total brain protein, making it a significant neuronal component

• UCHL1 variants have been linked to neurodegenerative disorders including Parkinson's and Alzheimer's diseases

• It serves as a marker for various cell types including Schwann Cells, Sympathetic Neurons, and Neuronal Cells

• The protein plays critical roles in neuronal health, protein degradation, and response to injury

Understanding UCHL1 function through antibody-based detection methods provides crucial insights into neuronal health, degeneration mechanisms, and potential therapeutic approaches for neurological disorders.

What cell types can be identified using UCHL1 antibodies?

UCHL1 antibodies serve as valuable markers for identifying specific cell populations in various tissue contexts. Research has established that UCHL1 antibodies can reliably identify:

• Schwann Cells - myelin-forming cells of the peripheral nervous system

• Lymphatic Vessel Endothelial Cells - cells lining lymphatic vessels

• Sympathetic Neurons - neurons of the sympathetic division of the autonomic nervous system

• Pancreatic Endocrine Cells - hormone-producing cells in the pancreas

• Neuronal Cells - cells of the central and peripheral nervous systems

When designing immunostaining protocols, researchers should optimize antibody dilution, incubation time, and detection methods based on the specific cell type being investigated. For dual or multiple labeling experiments, select antibodies raised in different host species to avoid cross-reactivity.

How do UCHL1 antibodies contribute to understanding neural stem cell (NSC) biology?

UCHL1 antibodies have proven instrumental in elucidating the relationship between UCHL1 and neural stem cell function. Recent research demonstrates that:

• UCHL1 facilitates Neural Stem Cell (NSC) activation by eliminating protein aggregates

• Upregulation of UCHL1 (using OE-UCHL1-LV) promotes NSC proliferation, which can be reversed by UCHL1 inhibitor LDN-57444

• Enhanced UCHL1 expression leads to increased differentiation into Tubulin β3+ neurons

• UCHL1 activates NSCs via UPP-dependent clearance of protein aggregates

To investigate this relationship experimentally, researchers can employ EdU incorporation assays to monitor NSC proliferation after UCHL1 modulation, use proteasome-specific affinity probes to measure proteasome activity, and quantify protein levels of proteasome 20S to assess UPS function. This methodological approach provides a comprehensive view of how UCHL1 influences neural stem cell biology and potential therapeutic applications in neurological conditions.

What are the best practices for validating UCHL1 antibody specificity?

Ensuring antibody specificity is critical for accurate experimental results. For UCHL1 antibodies, implement these validation methods:

• Western blot validation: Look for a single band at approximately 24.8 kDa corresponding to UCHL1

• Positive controls: Include samples known to express high levels of UCHL1 (neuronal tissues)

• Negative controls: Use tissues or cell lines with minimal UCHL1 expression

• Knockout/knockdown validation: Compare antibody staining in wild-type versus UCHL1 knockout models or siRNA-treated samples

• Cross-reactivity testing: Ensure the antibody doesn't recognize other UCH family members, particularly UCHL3 and UCHL5

When interpreting results, be cautious of signals at unexpected molecular weights and consider using secondary validation methods such as mass spectrometry to confirm antibody specificity. Multiple antibodies targeting different epitopes of UCHL1 can provide additional confidence in your findings.

How can UCHL1 antibodies be used to investigate the role of this protein in neurodegenerative disease models?

UCHL1 antibodies are powerful tools for examining how this protein contributes to neurodegenerative pathology. A comprehensive experimental approach should include:

• Localization studies: Use immunohistochemistry with UCHL1 antibodies to map protein distribution in healthy versus diseased brain tissues, noting changes in subcellular localization

• Protein interaction analysis: Employ co-immunoprecipitation with UCHL1 antibodies to identify binding partners in disease models

• Post-translational modification detection: Use modification-specific antibodies to identify altered states of UCHL1 in disease contexts

• Activity correlation: Combine antibody detection with functional assays using covalent activity-based probes such as IMP-1710

Research shows UCHL1 variants are linked with neurodegenerative disorders including Parkinson's and Alzheimer's diseases . Changes in UCHL1 expression, activity, or localization can be quantified using immunoblotting, activity-based protein profiling, and immunofluorescence respectively.

For comprehensive analysis, complement antibody-based approaches with genetic models such as the UCHL1 knockin mice (C152A mutation) that show attenuated gray and white matter injury and improved recovery of sensorimotor function after ischemic injury .

What methodological approaches can be used to study UCHL1 activity in intact cells using antibodies and small-molecule probes?

Investigating UCHL1 activity in intact cellular systems requires combining antibody-based detection with activity-sensitive probes. A sophisticated experimental workflow includes:

• Fluorescent activity-based probes: Use cell-permeable probes like 8RK59 to visualize and quantify active UCHL1 in live cells

• Covalent inhibitors and probes: Apply IMP-1710, which stereoselectively labels the catalytic cysteine of UCHL1 at low nanomolar concentrations in cells

• Complementary antibody detection: Follow probe labeling with antibody-based techniques to confirm UCHL1 specificity and quantify total protein levels

• Mutant controls: Include the significantly less active enantiomer IMP-1711 (compound 3) as a negative control to confirm specificity

The table below summarizes key UCHL1-specific probes and their properties:

For optimal results, combine activity-based profiling with antibody-based detection of total UCHL1 to calculate the ratio of active to total protein, providing insight into functional states rather than just expression levels.

How should researchers design experiments to study the impact of UCHL1 on protein aggregate clearance?

Investigating UCHL1's role in protein aggregate clearance requires a multifaceted experimental design. The following methodological approach is recommended:

• Modulation of UCHL1 levels:

  • Upregulate using overexpression vectors (e.g., OE-UCHL1-LV)

  • Downregulate using siRNA/shRNA or inhibitors like LDN-57444

  • Use UCHL1 knockout models as negative controls

• Assessment of aggregate clearance:

  • Quantify proteasome activity using proteasome-specific affinity probes

  • Measure protein levels of proteasome 20S as an indicator of UPS activation

  • Monitor aggregation-prone proteins using fluorescent tags or antibody detection

• Functional readouts:

  • For neural stem cells, assess proliferation via EdU incorporation assays

  • Examine cell fate determination by immunostaining for markers like Tubulin β3

  • Evaluate UPS-dependent aggregate clearance through biochemical fractionation

Research demonstrates that UCHL1 upregulation enhances proteasome activity, increases proteasome 20S levels, and facilitates the clearance of protein aggregates in neural stem cells . This activity directly correlates with improved neural stem cell activation and proliferation.

For comprehensive analysis, researchers should incorporate both biochemical assays (Western blots for ubiquitinated proteins) and imaging approaches (fluorescence microscopy of aggregate formation) across multiple timepoints to capture the dynamic nature of aggregate clearance.

What are the considerations when using UCHL1 antibodies to study its role in cerebral ischemia and recovery?

When investigating UCHL1's involvement in cerebral ischemia and recovery, researchers should implement a comprehensive experimental strategy with careful antibody selection:

• Model selection:

  • Use transient middle cerebral artery occlusion (MCAO) models

  • Consider UCHL1 C152A knockin mice that show resistance to ischemic injury

  • Include in vitro oxygen-glucose deprivation models for mechanistic studies

• Temporal dynamics:

  • Examine UCHL1 levels at multiple time points post-ischemia

  • Correlate UCHL1 expression/activity with injury progression and recovery phases

  • Use antibodies optimized for specific applications (IHC vs. Western blot)

• Functional assessments:

  • Evaluate gray and white matter injury using histological techniques

  • Assess sensorimotor function recovery through behavioral testing

  • Measure synaptic drive to pyramidal neurons and axonal conduction velocity

Research demonstrates that UCHL1 C152A knockin mice exhibit significantly attenuated gray and white matter injury following MCAO, with improved recovery of sensorimotor function compared to wild-type controls. By 21 days post-MCAO, these mice show recovered axonal conduction velocity, highlighting UCHL1's critical role in determining tissue survival and functional recovery .

For antibody-based experiments, researchers should select antibodies that recognize specific post-translational modifications of UCHL1 that occur during ischemic events, as these modifications may alter UCHL1 function and contribute to pathogenesis or recovery.

How can researchers combine UCHL1 antibodies with activity-based probes to comprehensively study UCHL1 biology?

To gain a complete understanding of UCHL1 biology, researchers should integrate antibody-based detection with activity-based profiling using this methodological framework:

• Complementary detection systems:

  • Use antibodies to quantify total UCHL1 protein levels

  • Apply activity-based probes like IMP-1710 to specifically label active UCHL1

  • Employ stereoselective inhibitors with appropriate controls (e.g., IMP-1711)

• Multi-dimensional analysis:

  • Correlate protein expression with activity levels across cellular compartments

  • Examine how post-translational modifications affect UCHL1 activity

  • Investigate how disease conditions alter the ratio of active to total UCHL1

• Technical integration:

  • For in vitro studies, rhodamine-tagged 9RK87 offers high potency

  • For cellular studies, cell-permeable probes like 8RK59 enable live imaging

  • Combine with UCHL1 antibodies in sequential labeling experiments

Recent research has developed cyanimide-containing inhibitors that act as irreversible binders, contradicting previous literature reports. The kinetics of these interactions (kobs/I = 7400-11000 M–1 s–1) reveal slow recovery of activity following dilution, consistent with slowly reversible inhibition .

Researchers should be aware that while antibodies detect all forms of UCHL1, activity-based probes selectively label catalytically active enzyme. This distinction enables discrimination between expression changes and functional alterations, providing deeper mechanistic insights than either approach alone.

What emerging applications of UCHL1 antibodies show the most promise for translational research?

UCHL1 antibodies demonstrate significant translational potential across multiple research domains. The most promising applications include:

• Neurodegenerative disease biomarkers:

  • UCHL1 antibodies can detect altered expression patterns in Parkinson's and Alzheimer's diseases

  • Changes in UCHL1 activity and post-translational modifications may serve as early disease indicators

• Cancer research and diagnostics:

  • High UCHL1 levels correlate with cancer progression and metastasis

  • Antibody-based detection could help stratify patients and predict outcomes

• Fibrosis treatment development:

  • UCHL1 is a potential therapeutic target in liver and lung fibrosis

  • Antibodies combined with activity assays can validate target engagement of novel inhibitors

• Neural regeneration therapies:

  • UCHL1's role in neural stem cell activation suggests therapeutic potential for neural repair

  • Antibodies can monitor treatment efficacy in spinal cord injury and stroke recovery models

The integration of antibody-based detection with functional assessments of UCHL1 activity provides a powerful approach for translational research. Particularly promising is the application of UCHL1 inhibitors like IMP-1710 that block pro-fibrotic responses in cellular models of idiopathic pulmonary fibrosis, supporting UCHL1 as a therapeutic target in fibrotic diseases .