USP36 Antibody, HRP conjugated

What is USP36 and why is it significant in deubiquitination research?

USP36 (Ubiquitin specific peptidase 36) is a member of the peptidase C19 family of deubiquitinating enzymes (DUBs). It plays crucial roles in multiple cellular processes including regulation of transcription, processing of rRNA, and modification of cellular proteins by removing ubiquitin. USP36 is also known by several synonyms including KIAA1453, FLJ12851, and DUB1 .

The protein has a molecular weight of approximately 123 kDa (calculated from its 1123 amino acid sequence), though it is typically observed at 100-150 kDa in Western blots due to post-translational modifications . USP36 is particularly significant in research because:

• It regulates nucleolar structure and function through deubiquitylation of proteins involved in ribosome biogenesis

• It plays essential roles in germline, epithelial, and intestinal stem cells

• It participates in neuronal precursor differentiation through interaction with TrkA and Nedd4-2

• It is involved in both chromatin organization and autophagy pathways

Research on USP36 contributes to our understanding of fundamental cellular processes and may provide insights into disease mechanisms where deubiquitination is dysregulated.

What is the subcellular localization of USP36 and how does this impact experimental design?

USP36 exhibits a complex subcellular distribution pattern that varies by cell type. Understanding its localization is critical for proper experimental design:

Research shows that while USP36 was initially described as primarily nucleolar, it is also present in non-nuclear compartments . In PC12-6/15 cells, fractionation experiments revealed that USP36 mainly accumulates in non-nuclear compartments, with partial co-localization with TrkA .

When designing experiments to study USP36, researchers should:

• Include multiple cellular fractions in their analysis

• Use co-localization studies with known markers for different compartments

• Consider cell type-specific differences in localization

• Employ proper controls for nuclear and cytoplasmic fractions (e.g., phospho-histone 3 for nuclear fractions; tubulin and GAPDH for non-nuclear fractions)

What are the key differences between HRP-conjugated and unconjugated USP36 antibodies?

HRP (horseradish peroxidase)-conjugated USP36 antibodies offer distinct advantages for certain applications compared to unconjugated versions:

HRP-conjugated USP36 antibodies like the OTI2F1 clone (Mouse Monoclonal, IgG1 isotype) are specifically designed for Western blot applications with a recommended dilution of 1:2000 . These conjugated antibodies eliminate the need for secondary antibody incubation, simplifying workflows and reducing background in certain applications.

Unconjugated versions, such as Proteintech's 68165-1-Ig mouse monoclonal, offer broader application ranges including Western Blot (1:2000-1:10000), Immunoprecipitation, Immunofluorescence (1:400-1:1600), and Flow Cytometry .

How should I optimize Western blot protocols specifically for USP36 detection using HRP-conjugated antibodies?

Optimizing Western blot protocols for USP36 detection requires careful consideration of several parameters:

Sample Preparation:

• Use appropriate lysis buffers containing protease inhibitors to prevent degradation of USP36 (122.7 kDa)

• Include phosphatase inhibitors if studying post-translational modifications

• For complete extraction, consider that USP36 is present in both nuclear and cytoplasmic fractions

Gel Selection and Transfer:

• Use 6-8% gels for optimal resolution of the high molecular weight USP36 (observed at 100-150 kDa)

• Extended transfer times (90-120 minutes) may be necessary for complete transfer of this large protein

Blocking and Antibody Incubation:

• For HRP-conjugated antibodies like OTI2F1 clone:

  • Use 5% non-fat dry milk or BSA in TBST for blocking (1 hour at room temperature)

  • Dilute antibody 1:2000 in blocking buffer

  • Incubate overnight at 4°C for optimal sensitivity

  • Include 0.05% sodium azide in the washing buffer to reduce background

Controls and Validation:

• Include positive control lysates (Jurkat, HeLa, HepG2, or U2OS cells show good endogenous expression)

• Consider knockdown/knockout controls to confirm specificity

• Use fractionation controls (phospho-histone 3 for nuclear fractions; tubulin and GAPDH for non-nuclear fractions)

Detection Optimization:

• For HRP-conjugated antibodies, select an appropriate substrate based on expected expression level

• Adjust exposure times carefully - USP36 may show multiple bands due to post-translational modifications or degradation

What are the critical parameters for generating and validating USP36 knockdown models?

Developing reliable USP36 knockdown models is essential for studying its function. Based on published methodologies:

Lentiviral shRNA Approach:

• Select appropriate shRNA sequences targeting USP36 (target validation data shows shRNA-1 and shRNA-6 achieved >70% knockdown efficiency, while shRNA-2, -3, and -4 were ineffective)

• Generate lentiviruses using HEK293FT cells transfected with:

  • 9 μg of pLVTHM control or USP36 shRNA plasmid

  • 6 μg of psPAX2 packaging plasmid

  • 5 μg of pMD.2G envelope plasmid

• Collect viral supernatant 48 hours post-transfection

• Process viral medium (centrifugation at 5000 rpm, filtration at 45 μm)

• Concentrate virus if needed (ultracentrifugation or LENTI-X solution)

• Monitor infection efficiency using GFP expression

• Validate knockdown efficiency 5-7 days post-infection

Validation Strategies:

• Western blot analysis using validated USP36 antibodies (recommended: 1:500-1:1000 dilution)

• qPCR to verify mRNA reduction

• Functional assays to confirm biological impact

• Assessment of nucleolar structure (a known consequence of USP36 depletion)

Considerations:

• Allow 5-7 days post-infection for efficient knockdown

• Include non-targeting shRNA controls

• Validate multiple shRNA constructs to control for off-target effects

• Consider rescue experiments with shRNA-resistant USP36 to confirm specificity

How can I verify USP36 interactions with binding partners using immunoprecipitation approaches?

Verifying USP36 protein-protein interactions requires careful experimental design. Based on published methodologies for studying USP36 interactions with TrkA and Nedd4-2:

Co-immunoprecipitation Protocol:

• Prepare cell lysates under conditions that preserve protein complexes:

  • Lyse cells in buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 10% glycerol, and protease inhibitors

  • Use gentle homogenization to preserve protein-protein interactions

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Immunoprecipitation:

  • Use 0.5-4.0 μg of anti-USP36 antibody per 1.0-3.0 mg of protein lysate

  • For HRP-conjugated antibodies, consider pre-treatment with a reducing agent to cleave disulfide bonds in HRP

  • Incubate with protein A/G beads for 2-4 hours or overnight at 4°C

  • Include IgG control immunoprecipitations

• Wash thoroughly (4-5 times) with buffer containing reduced detergent

• Elute and analyze by Western blotting:

  • Probe for USP36 to confirm successful IP

  • Probe for suspected interaction partners (e.g., TrkA, Nedd4-2)

  • Include input controls (5-10% of lysate used for IP)

Alternative Approaches:

• Reciprocal IP: Confirm interactions by immunoprecipitating the suspected binding partner (e.g., TrkA) and probing for USP36

• In vitro binding assays: Utilize GST-fusion proteins containing specific domains of interest (e.g., WW domains of Nedd4-2)

• Proximity ligation assay: For detecting endogenous protein interactions in situ

Controls and Validation:

• Include stimulation conditions where relevant (e.g., NGF treatment for TrkA studies)

• Use domain deletion mutants to map interaction regions

• Consider competition assays to test binding specificity (e.g., USP36 competition with TrkA for binding to Nedd4-2)

What methods are optimal for studying USP36 deubiquitinating activity in vitro?

USP36 deubiquitinating activity can be assessed using several complementary approaches:

In Vitro Ubiquitination/Deubiquitination Assay:

• Immunoprecipitate purified TrkA and FLAG-HA-USP36 from HEK293 cell lysates

• Elute USP36 using FLAG peptide

• Prepare reaction mixture containing:

  • Immunoprecipitated TrkA

  • GST or GST-WW3-4HECT E3 ligase

  • FLAG-HA-USP36 (variable amounts)

  • E1 (rabbit, 150 ng)

  • UbcH5b (300 ng)

  • Buffer: 25 mM Tris-HCl pH 7.5, 120 mM NaCl, 2 mM MgCl₂, 2 mM ATP, 500 μM DTT

  • Bovine ubiquitin (500 ng/μl)

• Incubate for 2 hours at 30°C

• Stop reaction with 2× SDS buffer

• Analyze by SDS-PAGE and Western blotting

Analysis of Substrate Ubiquitination:

• Transfect cells with constructs expressing USP36 (or siRNA for knockdown)

• Immunoprecipitate potential substrates (e.g., TrkA)

• Probe for ubiquitin to assess changes in ubiquitination status

• Include controls:

  • Catalytically inactive USP36 mutant

  • Proteasome inhibitors (MG132) to prevent degradation of ubiquitinated proteins

Fluorescent Ubiquitin Substrates:

• Use commercially available fluorogenic ubiquitin substrates to measure deubiquitinating activity

• Compare wild-type USP36 activity to catalytically inactive mutants

• Determine enzyme kinetics (Km, Vmax)

How can I troubleshoot non-specific binding or high background when using HRP-conjugated USP36 antibodies?

Non-specific binding and high background are common challenges when working with antibodies. For HRP-conjugated USP36 antibodies:

Common Issues and Solutions:

Specific Strategies for HRP-Conjugated Antibodies:

• Include reducing agents in sample buffer to prevent HRP dimers

• Consider adding 0.05% Tween-20 to antibody dilution buffer to reduce non-specific binding

• For Western blots, try alternative membrane blocking (SuperBlock, StartingBlock)

• Ensure HRP activity using a test substrate before main experiment

• Use mild stripping and reprobing to confirm band specificity

What are the critical factors to consider when using USP36 antibodies across different species?

Cross-species reactivity is an important consideration when selecting USP36 antibodies for comparative studies:

Species Reactivity Data:

Sequence Homology Considerations:

• Human USP36 shows high conservation with mouse and rat orthologs

• Mouse USP36 is 1109 amino acids with 88% identity to human

• The immunogen region (amino acids 589-972 of human USP36) used for OTI2F1 clone should be evaluated for conservation across target species

Validation Strategies for Cross-Species Use:

• Perform Western blot analysis on lysates from multiple species

• Include positive controls from well-characterized cell lines

• Validate using knockdown/knockout samples when available

• Consider epitope mapping to identify species-specific differences

• When using antibodies outside their validated species, perform titration experiments to optimize conditions

Technical Recommendations:

• For mouse monoclonal antibodies used in mouse tissues, use Mouse-on-Mouse detection systems to reduce background

• For rat samples, evaluate background with secondary-only controls

• Consider the specific application requirements (WB vs. IF vs. IHC)

• When comparing expression across species, use multiple antibodies targeting different epitopes

How can USP36 antibodies be effectively employed in studying nucleolar stress responses?

USP36 plays critical roles in nucleolar function and ribosome biogenesis, making it valuable for nucleolar stress studies:

Immunofluorescence Protocol for Nucleolar Stress:

• Culture cells on poly-D-lysine coated coverslips (1 mg/ml)

• Induce nucleolar stress using established methods:

  • Low-dose actinomycin D (5 nM, 3-6 hours)

  • 5-FU treatment

  • Serum starvation

  • Knockdown of ribosomal proteins

• Fix cells with 4% paraformaldehyde with 20% sucrose in PBS (5 minutes)

• Quench with 50 mM NH₄Cl (10 minutes)

• Block in PBS with 10% FBS, 2% BSA, 0.1% Tween 20, and 0.1% Triton X-100 (1 hour)

• Incubate with primary antibodies:

  • Anti-USP36 (1:200-1:800 dilution)

  • Co-stain with nucleolar markers (fibrillarin, nucleolin)

• Apply fluorescent secondary antibodies (40 minutes)

• Mount in Prolong Gold medium

• Image using confocal microscopy

Analysis Parameters:

• Quantify USP36 nucleolar-to-nucleoplasmic ratio

• Measure co-localization with nucleolar markers

• Assess nucleolar morphology changes

• Correlate with rRNA transcription/processing markers

Complementary Approaches:

• Chromatin immunoprecipitation to analyze USP36 association with rDNA

• RNA immunoprecipitation to study USP36 interactions with rRNA processing factors

• Proximity ligation assays to detect protein-protein interactions in nucleoli

• Live-cell imaging using fluorescently-tagged USP36 to monitor dynamic responses

What are the best approaches for studying the role of USP36 in TrkA signaling pathways using HRP-conjugated antibodies?

USP36 interacts with TrkA and affects its activation kinetics , suggesting important roles in neurotrophin signaling:

Experimental Design for TrkA Signaling Studies:

• Time-course activation studies:

  • Treat neuronal cells (PC12-6/15) with NGF (50-100 ng/ml)

  • Collect samples at multiple timepoints (0, 5, 15, 30, 60 minutes)

  • Use phospho-specific antibodies to monitor TrkA activation

  • Compare wild-type vs. USP36 knockdown conditions

• TrkA ubiquitination analysis:

  • Immunoprecipitate TrkA from control and USP36-depleted cells

  • Probe for ubiquitin to assess ubiquitination status

  • Use HRP-conjugated secondary antibodies for detection

  • Consider K48 vs. K63 linkage-specific ubiquitin antibodies to determine degradative vs. signaling roles

• Signaling pathway analysis:

  • Monitor downstream effectors (Erk1/2, Akt, PLCγ)

  • Use well-characterized phospho-specific antibodies

  • Compare kinetics and magnitude of activation

• Competition assays:

  • Assess how USP36 affects TrkA-Nedd4-2 interactions

  • Perform in vitro binding assays with purified components

  • Use GST-WW domain pulldowns with variable amounts of USP36

Controls and Validations:

• Include USP36 catalytic mutants to distinguish between scaffolding and enzymatic functions

• Use Nedd4-2 C962S catalytically inactive mutant as control

• Compare effects of USP36 vs. other deubiquitinating enzymes

• Consider dose-response studies with varying NGF concentrations

How can I integrate USP36 studies into broader deubiquitinase research programs?

USP36 research can be integrated into broader DUB studies through systematic approaches:

Comparative DUB Analysis Framework:

• Substrate profiling:

  • Develop systematic approaches to identify USP36 substrates

  • Compare against substrates of related DUBs

  • Use proteomics approaches (SILAC, TMT) to identify changes in the ubiquitinome

  • Focus on nucleolar and ribosomal biogenesis proteins

• Structural and functional classification:

  • Position USP36 within the USP family based on sequence, domain architecture, and substrate specificity

  • Compare catalytic mechanisms and regulatory domains

  • Assess evolutionary conservation across species

• Inhibitor screening and development:

  • Develop assays suitable for high-throughput screening

  • Test panels of DUB inhibitors for selectivity against USP36

  • Conduct structure-activity relationship studies

  • Validate hits with orthogonal assays

• Genetic interaction mapping:

  • Perform CRISPR screens in USP36-depleted backgrounds

  • Identify synthetic lethal interactions

  • Map genetic dependencies in different cellular contexts

  • Compare with other DUB family members

Integration with Multi-omics Approaches:

• Transcriptomics to identify gene expression changes upon USP36 manipulation

• Proteomics to map the USP36 interactome across cellular compartments

• Ubiquitinomics to catalog USP36-dependent ubiquitination events

• Systems biology approaches to position USP36 within ubiquitin-dependent cellular networks