USP20 Antibody, FITC conjugated

What is USP20 and why is it an important research target?

USP20 (ubiquitin specific peptidase 20) is a deubiquitinating enzyme that plays critical roles in multiple cellular pathways. Also known as VDU2 (VHL-interacting deubiquitinating enzyme 2), KIAA1003, or LSFR3A, USP20 functions as a regulator of G-protein coupled receptor signaling by mediating the deubiquitination of beta-2 adrenergic receptor (ADRB2) . It also deubiquitinates DIO2, regulating thyroid hormone metabolism, and stabilizes HIF1A through deubiquitination . Importantly, USP20 acts as a positive regulator of autophagy initiation through stabilization of ULK1 . These diverse functions make USP20 a significant target for research in multiple disease contexts including cancer, metabolic disorders, and neurological conditions.

What are the typical applications for FITC-conjugated USP20 antibodies?

FITC-conjugated USP20 antibodies are primarily utilized in fluorescence-based detection methods. While standard USP20 antibodies are commonly used in Western Blot (1:500-1:1000 dilution), Immunoprecipitation (0.5-4.0 μg for 1.0-3.0 mg of protein lysate), and Immunohistochemistry (1:50-1:500 dilution) , FITC-conjugated variants are specifically optimized for:

• Immunofluorescence microscopy for cellular localization studies

• Flow cytometry for quantitative analysis of USP20 expression

• ELISA for quantitative protein detection

The FITC conjugation eliminates the need for secondary antibodies in fluorescence-based applications, streamlining experimental workflows and reducing background signal.

What species reactivity can be expected with commercially available USP20 antibodies?

Based on the search results, commercially available USP20 antibodies show reactivity with:

When selecting an antibody, researchers should verify species reactivity for their specific model system. Cross-reactivity testing may be necessary when working with species not listed in the manufacturer's specifications .

How should USP20 antibodies be stored to maintain optimal activity?

For maximum stability and activity retention, USP20 antibodies should be stored according to these guidelines:

• Store at -20°C for long-term storage

• Antibodies are typically stable for one year after shipment when stored properly

• Aliquoting is recommended to avoid repeated freeze/thaw cycles, although some formulations specifically state that aliquoting is unnecessary for -20°C storage

• USP20 antibodies are commonly supplied in PBS buffer containing preservatives such as 0.02-0.09% sodium azide and may contain 50% glycerol (pH 7.3)

• Some smaller volume preparations (20μl) may contain 0.1% BSA as a stabilizer

Proper storage significantly impacts experimental reproducibility and sensitivity in applications like Western blotting and immunofluorescence.

How can researchers validate the specificity of USP20 antibodies in deubiquitination studies?

Validating USP20 antibody specificity in deubiquitination studies requires multiple complementary approaches:

• Genetic validation: Use USP20 knockdown or knockout models as negative controls. The search results indicate that several publications have used siRNA-mediated knockdown of USP20 to confirm antibody specificity and demonstrate its functional role .

• Recombinant protein controls: Utilize purified USP20 wild-type (WT) versus catalytically inactive (CI) proteins in in vitro deubiquitination assays. As described in search result , these can be isolated from HEK293 cells transiently expressing Flag-USP20-WT or Flag-USP20-CI.

• Substrate-specific validation: For studying USP20's deubiquitinating activity on specific substrates like ULK1, researchers can perform an in vitro deubiquitination assay where purified Flag-ULK1 is incubated with purified USP20-WT or USP20-CI proteins in deubiquitination buffer (40 mM Tris, 50 mM NaCl, 5 mM MgCl₂, 5 mM dithiothreitol, 2 mM ATP, pH 7.6) at 37°C for 6 hours .

• Immunoprecipitation validation: Confirm interaction between USP20 and its substrates using endogenous immunoprecipitation, as demonstrated by the interaction between USP20 and ULK1 in search result .

What methodological considerations are important when using FITC-conjugated USP20 antibodies for co-localization studies?

When conducting co-localization studies with FITC-conjugated USP20 antibodies, researchers should implement these methodological controls:

• Spectral considerations: FITC emits green fluorescence (λ emission ~520 nm), so select complementary fluorophores with minimal spectral overlap for multi-color imaging.

• Fixation optimization: Different fixation methods may affect epitope accessibility and FITC signal intensity. Test multiple fixation protocols (4% paraformaldehyde, methanol, or acetone) to determine optimal conditions for USP20 detection.

• Autofluorescence mitigation: Tissues with high autofluorescence in the green spectrum may complicate FITC-based detection. Consider using Sudan Black B (0.1-0.3%) treatment post-fixation to reduce autofluorescence.

• Quantitative co-localization analysis: Use appropriate software tools (ImageJ with Coloc2, CellProfiler) and statistical methods (Pearson's correlation coefficient, Manders' overlap coefficient) to quantify co-localization beyond visual assessment.

• Control for non-specific binding: Include appropriate negative controls (isotype control antibodies with FITC conjugation) and blocking steps to minimize non-specific fluorescence.

How can researchers optimize immunoprecipitation protocols using USP20 antibodies to study deubiquitination mechanisms?

Optimizing immunoprecipitation (IP) for studying USP20-mediated deubiquitination requires careful protocol adjustments:

• Lysis buffer optimization: Use a lysis buffer specifically designed to preserve deubiquitinase activity while efficiently extracting USP20. The search results indicate successful IP using buffer containing 0.4% NP-40, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 2 mM EDTA, 10 mM NaF, 1 mM Na₃VO₄, and protease inhibitor cocktail .

• Incubation conditions: Optimal conditions include incubation with specific USP20 antibodies at 4°C for 12 hours followed by 1-hour incubation with protein G agarose beads .

• Preserving ubiquitination status: Add deubiquitinase inhibitors (e.g., N-ethylmaleimide) and proteasome inhibitors (e.g., MG132) to lysis buffers when studying ubiquitinated substrates.

• Antibody amount optimization: For USP20 IP, a recommended range of 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate provides optimal results .

• Sequential IP approach: For studying USP20 interaction with specific substrates like ULK1, consider a sequential IP approach to first pull down the substrate and then detect USP20, or vice versa.

What are the experimental considerations when comparing results between different detection methods using USP20 antibodies?

When comparing results from different detection methods, researchers should account for the following variables:

• Epitope accessibility variations: The USP20 epitope accessibility varies between applications. For example, the Proteintech antibody (17491-1-AP) targets a fusion protein immunogen , while the Abbexa antibody targets a KLH-conjugated synthetic peptide between amino acids 310-339 from the central region of human USP20 .

• Native versus denatured states: Western blotting detects denatured USP20 (~110-120 kDa observed molecular weight) , while applications like IP and IF detect native conformations, potentially yielding different results.

• Sensitivity thresholds: Different detection methods have varying sensitivity thresholds:

  • Western blotting: Sensitive for abundant proteins

  • Immunofluorescence: Higher sensitivity for localized concentrations

  • ELISA: Highest quantitative sensitivity

• Quantification methods: Each technique requires specific quantification approaches:

  • For WB: Densitometry normalization to loading controls

  • For IF: Mean fluorescence intensity measurements

  • For ELISA: Standard curve calculations

• Cross-validation strategy: Important findings should be validated using at least two independent detection methods and ideally with antibodies targeting different epitopes.

What are common sources of non-specific binding with USP20 antibodies and how can they be mitigated?

Non-specific binding can significantly impact experimental results. Common sources and mitigation strategies include:

• Cross-reactivity with related DUBs: USP20 belongs to the USP family, which has over 50 members with conserved catalytic domains. To mitigate:

  • Use antibodies targeting unique regions outside the catalytic domain

  • Include USP20 knockout/knockdown controls to confirm specificity

  • Pre-absorb antibodies with recombinant proteins from closely related USP family members

• Inadequate blocking: Optimize blocking conditions using 5% non-fat dry milk or BSA in TBST/PBST. For tissues with high background, consider specialized blocking reagents containing normal serum from the same species as the secondary antibody.

• Fixation artifacts: Different fixation methods can create epitope masking or exposure. The search results suggest using TE buffer pH 9.0 for antigen retrieval in IHC applications, with citrate buffer pH 6.0 as an alternative .

• Sample preparation issues: Incomplete denaturation for WB can cause aggregation and smearing. Ensure thorough sample preparation using appropriate buffers and denaturation conditions.

• Antibody concentration optimization: Titrate antibody concentration for each application. The recommended dilutions vary significantly between applications (1:500-1:1000 for WB vs. 1:50-1:500 for IHC) .

How can researchers troubleshoot discrepancies in USP20 molecular weight observed across different experimental systems?

The calculated molecular weight of USP20 is 102 kDa (914 amino acids), but the observed molecular weight in experimental systems typically ranges from 110-120 kDa . This discrepancy can be attributed to several factors:

• Post-translational modifications: USP20 undergoes modifications that affect migration on SDS-PAGE:

  • Phosphorylation sites have been identified on USP20

  • Potential glycosylation may occur

  • USP20 itself is regulated by ubiquitination

• Isoform variations: Multiple splice variants of USP20 exist. Verify which isoform is predominant in your experimental system through RT-PCR or sequencing.

• Sample preparation effects: Different lysis and denaturation methods can affect observed molecular weight:

  • Use freshly prepared samples to minimize degradation

  • Include appropriate protease inhibitors

  • Consider native versus reducing conditions

• Gel percentage optimization: Use gradient gels (4-12% or 4-15%) for better resolution of high molecular weight proteins.

• Molecular weight markers: Use high-quality, validated protein markers appropriate for the expected weight range.

What methodological approaches can address inconsistencies in co-immunoprecipitation results with USP20 antibodies?

When facing inconsistent co-immunoprecipitation results with USP20 antibodies, consider these methodological refinements:

• Cross-linking optimization: Implement reversible cross-linking with DSP (dithiobis[succinimidyl propionate]) to stabilize transient protein interactions before cell lysis.

• Buffer composition adjustment: The search results indicate successful use of lysis buffer containing 0.4% NP-40, 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 2 mM EDTA, 10 mM NaF, 1 mM Na₃VO₄, and protease inhibitors . Adjust salt concentration (125-150 mM NaCl) to optimize interaction stringency.

• Antibody orientation alternatives: Try both orientations of the co-IP:

  • IP with anti-USP20 and blot for interacting partners

  • IP with antibodies against suspected interacting partners and blot for USP20

• Pre-clearing strategy: Implement rigorous pre-clearing of lysates with protein G beads alone to reduce non-specific binding.

• Endogenous versus overexpression systems: Compare results between endogenous co-IP and overexpression systems. The search results demonstrate successful endogenous interactions between USP20 and ULK1 .

How can FITC-conjugated USP20 antibodies be utilized in live-cell imaging to study dynamic protein interactions?

FITC-conjugated USP20 antibodies can be adapted for live-cell imaging through these methodological approaches:

• Cell-permeable antibody derivatives: Consider using:

  • Electroporation-mediated antibody delivery

  • Protein transfection reagents (Chariot, BioPORTER)

  • Single-chain variable fragments (scFv) with cell-penetrating peptides

• Complementary fluorescent protein approaches: Combine antibody studies with expression of fluorescently tagged USP20 binding partners to visualize interactions in real-time.

• Pulse-chase methodology: Implement pulse-chase labeling techniques to monitor USP20 dynamics following stimulation of pathways known to involve USP20, such as beta-2 adrenergic receptor signaling.

• Photobleaching studies: Use FRAP (Fluorescence Recovery After Photobleaching) to assess USP20 mobility and dynamic interactions with substrates.

• Environmental considerations: FITC fluorescence is pH-sensitive, which can be leveraged to study USP20 translocation between cellular compartments with different pH environments.

What are effective strategies for studying the role of USP20 in autophagic pathways using immunofluorescence techniques?

To effectively study USP20's role in autophagy using immunofluorescence:

• Co-localization with autophagy markers: Use FITC-conjugated USP20 antibodies in combination with antibodies against autophagy markers:

  • LC3B for autophagosome formation

  • LAMP1 for autolysosome fusion

  • ULK1 (a direct USP20 substrate in autophagy regulation)

• Autophagy induction protocols: Establish standardized protocols for autophagy induction:

  • Nutrient starvation (EBSS medium, serum starvation)

  • mTOR inhibition (rapamycin, Torin1)

  • Chemical inducers (trehalose, SMER compounds)

• Temporal analysis: Implement time-course experiments to capture the dynamic association of USP20 with autophagy components during initiation, elongation, and maturation phases.

• Quantitative analysis methods: Apply automated image analysis for quantification:

  • Puncta counting for autophagosomes

  • Co-localization coefficients for USP20 with ULK1 and other proteins

  • Fluorescence intensity measurements across cellular compartments

• Functional validation: Combine imaging with functional readouts:

  • LC3-II/LC3-I conversion by Western blot

  • Autophagic flux assays with bafilomycin A1

  • Selective substrate degradation assays

How can researchers integrate USP20 antibody-based detection methods into multi-omics approaches for comprehensive pathway analysis?

Integrating USP20 antibody-based detection into multi-omics research requires strategic methodological planning:

• Sequential sample utilization:

  • Split biological samples for parallel proteomics, transcriptomics, and antibody-based analyses

  • Use consecutive tissue sections for spatial transcriptomics and immunohistochemistry

  • Implement microscopy-to-mass spectrometry workflows for spatial proteomics validation

• Correlation analysis frameworks:

  • Develop computational pipelines to correlate USP20 protein levels/localization with transcriptomic data

  • Implement network analysis to position USP20 within broader signaling networks

  • Apply machine learning approaches to identify patterns across multi-omics datasets

• Validation strategies:

  • Confirm USP20-substrate interactions identified in proteomics using co-immunoprecipitation

  • Validate changes in USP20 localization or expression using immunofluorescence or Western blotting

  • Use CRISPR/Cas9 editing to confirm functional roles predicted by multi-omics analyses

• Temporal dimension integration:

  • Design time-course experiments spanning all omics platforms

  • Create integrated visualization tools for temporal data presentation

  • Develop mathematical models incorporating temporal dynamics

• Single-cell adaptations:

  • Combine single-cell RNA-seq with imaging mass cytometry using USP20 antibodies

  • Implement in situ proximity ligation assays for detecting USP20-substrate interactions at single-cell resolution

What are the latest methodological advances in studying USP20's role in beta-2 adrenergic receptor recycling?

Recent methodological advances for studying USP20's role in ADRB2 recycling include:

• Live-cell ubiquitination sensors: Implementation of FRET-based ubiquitin sensors to monitor real-time deubiquitination of ADRB2 by USP20 following receptor stimulation.

• Trafficking analysis techniques: Application of pH-sensitive fluorescent probes to track receptor movement through endocytic compartments in the presence and absence of USP20 activity.

• Microfluidic approaches: Development of microfluidic platforms allowing precise temporal control of agonist exposure while simultaneously monitoring USP20-ADRB2 interactions.

• Super-resolution microscopy: Implementation of techniques like STORM and PALM to visualize USP20-mediated deubiquitination events at the plasma membrane and endosomal compartments with nanometer precision.

• Ubiquitin chain-specific analysis: Utilization of ubiquitin chain-specific antibodies to determine how USP20 differentially affects 'Lys-48'- versus 'Lys-63'-linked polyubiquitin chains on ADRB2, as the search results indicate USP20 can mediate deubiquitination of both chain types .

How can researchers effectively study the interplay between USP20 and HIF1A stabilization in cancer models?

To effectively study USP20-HIF1A interactions in cancer contexts:

• 3D culture systems: Implement spheroid or organoid models that better recapitulate tumor hypoxic microenvironments where HIF1A stabilization is physiologically relevant.

• Oxygen gradient systems: Utilize specialized culture systems with controlled oxygen gradients to study USP20-mediated HIF1A stabilization under varying hypoxic conditions.

• Patient-derived xenograft (PDX) models: Establish PDX models to study USP20-HIF1A dynamics in more clinically relevant systems, using USP20 antibodies validated for both human and mouse reactivity .

• CRISPR-based approaches: Implement CRISPR-Cas9 gene editing to:

  • Create USP20 catalytic mutants to distinguish between scaffolding and enzymatic functions

  • Introduce targeted mutations in the USP20-HIF1A interaction interface

  • Develop cellular systems with endogenously tagged USP20 and HIF1A

• Selective inhibitor studies: Apply emerging small molecule USP20 inhibitors in combination with antibody-based detection methods to dissect the functional impact of USP20 inhibition on HIF1A levels and activity in cancer models.

What methodological considerations are important when investigating USP20's role in autophagy regulation through ULK1 stabilization?

When investigating USP20's role in autophagy via ULK1 stabilization, researchers should implement these methodological considerations:

• Conditional expression systems: Establish tetracycline-inducible or similar systems to control USP20 expression levels with temporal precision.

• ULK1 half-life measurements: Implement cycloheximide chase assays with and without USP20 expression/activity to measure ULK1 protein stability, as demonstrated in the search results .

• Ubiquitination site mapping: Employ mass spectrometry-based approaches to identify specific lysine residues on ULK1 that are deubiquitinated by USP20.

• Autophagy flux measurements: Combine ULK1 stabilization studies with downstream autophagy flux measurements:

  • LC3 turnover assays with and without lysosomal inhibitors

  • Long-lived protein degradation assays

  • Selective autophagy substrate clearance (e.g., p62/SQSTM1)

• In vitro reconstitution: Develop in vitro deubiquitination assays using purified components as described in the search results: purified Flag-ULK1 incubated with purified USP20 wild-type (WT) or catalytically inactive (CI) proteins in deubiquitination buffer (40 mM Tris, 50 mM NaCl, 5 mM MgCl₂, 5 mM dithiothreitol, 2 mM ATP, pH 7.6) for 6 hours at 37°C .