USP6 Antibody

What is USP6 and what are its primary functions in cellular biology?

USP6 (also known as Tre-2 or Ubiquitin-Specific Peptidase 6) is a deubiquitinating enzyme with ATP-independent isopeptidase activity that cleaves at the C-terminus of ubiquitin moieties . This 1,406 amino acid protein is characterized by its hydrophilic nature and contains two charge clusters indicating potential nucleic acid-binding regions .

USP6 participates in several critical cellular processes:

• Self-deubiquitination catalysis

• Promotion of plasma membrane localization of ARF6

• Selective regulation of ARF6-dependent endocytic protein trafficking

• Initiation of tumorigenesis by inducing matrix metalloproteinase production via NF-kappa-B activation

• Modulation of Wnt signaling pathways through deubiquitylation of Frizzled receptors

Notably, USP6 originated from a chimeric fusion of two genes (USP32 and TBC1D3) and exists exclusively in the hominoid lineage of primates, suggesting evolutionary significance potentially related to speciation .

How do I select the appropriate USP6 antibody for my specific research application?

Selection of an appropriate USP6 antibody should be guided by your experimental requirements:

When selecting, consider:

• Species reactivity: Many USP6 antibodies detect human, mouse, and rat proteins, but verify cross-reactivity

• Epitope location: For domain-specific studies, select antibodies raised against relevant protein regions (e.g., within aa 1100-1400 for C-terminal studies)

• Validation status: Prioritize antibodies with published validation data or those cited in peer-reviewed publications

What are the key differences between polyclonal and monoclonal USP6 antibodies?

Polyclonal USP6 Antibodies:

• Recognize multiple epitopes within the USP6 protein

• Typically provide stronger signals due to binding multiple sites

• Examples include rabbit polyclonal antibodies that recognize regions within amino acids 1100-1400

• Often suitable for applications like Western blot and immunohistochemistry

• May show higher batch-to-batch variability

Monoclonal USP6 Antibodies:

• Recognize a single epitope with high specificity

• Provide consistent results with minimal batch-to-batch variation

• Examples include mouse monoclonal IgG1 kappa light chain antibody (D-11)

• Available in both unconjugated and conjugated forms (HRP, PE, FITC, Alexa Fluor)

• May be less sensitive but more specific than polyclonal antibodies

The choice between monoclonal and polyclonal depends on your experimental goals—use monoclonal when high specificity is required and polyclonal when signal amplification is needed.

What are the optimal protocols for Western blot detection of USP6?

Sample Preparation:

• Prepare cell or tissue lysates using RIPA buffer supplemented with protease inhibitors

• Determine protein concentration using Bradford or BCA assay

• Load 20-50 μg of total protein per lane

Protocol Optimization:

• Primary Antibody: Use rabbit polyclonal USP6 antibody at 1/500 dilution or mouse monoclonal at manufacturer's recommended concentration

• Expected Molecular Weight: ~140 kDa for full-length USP6 protein

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour

• Membrane Washing: 3-5 washes with TBST between antibody incubations

• Secondary Antibody: HRP-conjugated anti-rabbit or anti-mouse IgG (depending on primary)

Troubleshooting Tips:

• For weak signals: Increase protein loading or antibody concentration, extend exposure time

• For high background: Increase washing steps, decrease antibody concentration, optimize blocking

• For multiple bands: Verify with positive control, consider using mouse monoclonal D-11 antibody for higher specificity

How should I optimize immunohistochemistry protocols for USP6 detection in different tissue types?

Tissue Processing Recommendations:

• Fix tissues in 10% neutral buffered formalin for 24-48 hours

• Process and embed in paraffin following standard protocols

• Section at 4-5 μm thickness

Antigen Retrieval Methods:

• Heat-Induced Epitope Retrieval (HIER): Use citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Pressure cooker method: 125°C for 30-40 seconds or 95°C for 20 minutes

• Enzymatic retrieval may not be recommended for USP6 detection

Staining Protocol Optimization:

• Primary Antibody: Dilute rabbit polyclonal antibody (1:100-1:200) or mouse monoclonal antibody according to datasheet recommendations

• Incubation: Overnight at 4°C or 1 hour at room temperature

• Detection System: Polymer-based detection systems generally produce better results than avidin-biotin methods

• Counterstain: Hematoxylin for nuclear visualization

Controls and Validation:

• Positive Control: Include tissues known to express USP6 (certain neoplasms with USP6 translocation)

• Negative Control: Omit primary antibody or use isotype control

• Blocking: Include appropriate blocking steps to minimize non-specific binding

What are the critical considerations for immunofluorescence studies using USP6 antibodies?

Sample Preparation:

• For cultured cells: Fix with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 in PBS for 5-10 minutes

• Block with 1-5% normal serum (from secondary antibody host species) for 30-60 minutes

Protocol Optimization:

• Primary Antibody: Use rabbit polyclonal or mouse monoclonal antibody at recommended dilutions

• Incubation: 1-2 hours at room temperature or overnight at 4°C

• Secondary Antibody: Fluorophore-conjugated antibodies (e.g., Alexa Fluor series) at 1:500-1:1000

• Nuclear Counterstain: DAPI (1 μg/ml) for 5 minutes

Co-localization Studies:

• For dual staining, select USP6 antibodies from different host species than your second target

• Consider directly conjugated USP6 antibodies (FITC, PE) to eliminate cross-reactivity

• Include appropriate controls for fluorescence bleed-through

Image Acquisition Guidelines:

• Use sequential scanning for multi-channel confocal microscopy

• Optimize exposure settings to prevent saturation

• Capture Z-stacks for 3D visualization of subcellular localization

How can USP6 antibodies be utilized to investigate its role in oncogenic signaling pathways?

USP6 has been implicated in tumorigenesis through multiple mechanisms, including NF-κB activation and Wnt signaling modulation . Advanced research approaches include:

Chromatin Immunoprecipitation (ChIP) Analysis:

• Use USP6 antibodies to immunoprecipitate USP6-bound chromatin

• Analyze promoter regions of NF-κB target genes to assess transcriptional regulation

• Combine with sequencing (ChIP-seq) for genome-wide binding analysis

Proximity Ligation Assay (PLA):

• Employ USP6 antibodies together with antibodies against suspected interaction partners

• Visualize protein-protein interactions in situ with single-molecule resolution

• Quantify interaction events in different cellular compartments or treatment conditions

USP6 Deubiquitinase Activity Assays:

• Immunoprecipitate USP6 using specific antibodies (e.g., USP6/32 D-11)

• Measure deubiquitinase activity using fluorogenic substrates

• Assess effects of inhibitors or mutations on enzymatic function

Wnt Signaling Analysis:

• Use USP6 antibodies for co-immunoprecipitation with Frizzled receptors

• Examine β-catenin stabilization and nuclear translocation following USP6 modulation

• Analyze TOP/FOP reporter activity in cells with altered USP6 expression

What techniques can be employed to study USP6's evolutionary uniqueness in hominoid primates?

USP6 represents a fascinating case study in evolutionary biology as a chimeric gene exclusive to hominoids . Advanced research approaches include:

Comparative Immunohistochemistry:

• Use cross-reactive USP6 antibodies to compare expression patterns across primate tissues

• Focus on testis samples, as USP6 shows testis-specific expression

• Correlate expression patterns with phylogenetic relationships

Domain-Specific Antibody Applications:

• Utilize antibodies specific to USP32- and TBC1D3-derived domains of USP6

• Compare relative conservation of these domains across hominoid species

• Identify potential selective pressures on different protein regions

Reproductive Barrier Analysis:

• Investigate USP6 localization in reproductive tissues using immunofluorescence

• Compare with non-hominoid primate controls lacking USP6

• Correlate findings with reproductive compatibility data between species

Genomic Database Integration:

• Combine antibody-based protein expression data with genomic information

• Analyze USP6 sequence variations across hominoid species

• Develop evolutionary models for USP6 emergence and selection

How can researchers investigate the relationship between USP6 and ARF6-dependent trafficking?

USP6 promotes plasma membrane localization of ARF6 and regulates ARF6-dependent endocytic protein trafficking . Advanced methodological approaches include:

Live Cell Imaging with Fluorescently Tagged Proteins:

• Co-express fluorescently tagged USP6 and ARF6

• Use immunofluorescence to validate expression with anti-USP6 antibodies

• Track trafficking events in real-time under various conditions

Endosomal Fractionation and Analysis:

• Perform subcellular fractionation to isolate endosomal compartments

• Use USP6 antibodies for Western blot analysis of fractions

• Co-localize USP6 with endosomal markers and ARF6

Protein-Protein Interaction Studies:

• Conduct co-immunoprecipitation using USP6 antibodies to pull down ARF6

• Perform reverse co-IP with ARF6 antibodies to validate interaction

• Map interaction domains using truncated protein constructs

Functional Trafficking Assays:

• Monitor internalization of labeled receptors in cells with modulated USP6 levels

• Use USP6 antibodies to correlate trafficking defects with USP6 localization

• Employ super-resolution microscopy for detailed visualization of trafficking events

How should I interpret contradictory results between different USP6 antibody clones?

Contradictory results between different USP6 antibody clones are not uncommon and may arise from several factors:

Potential Causes of Discrepancies:

• Different epitope recognition: Antibodies targeting different regions of USP6 may yield varying results

• Isoform specificity: Some antibodies may preferentially detect certain USP6 isoforms (e.g., isoform 2 shows deubiquitinating activity while isoform 3 does not)

• Post-translational modifications: Modifications may mask epitopes recognized by certain antibodies

• Cross-reactivity: Some antibodies may cross-react with related proteins like USP32

Systematic Resolution Approach:

• Epitope Mapping Analysis:

  • Compare the immunogens used to generate each antibody (e.g., aa 1100-1400 for ab224725)

  • Consider whether the recognized epitopes might be affected by experimental conditions

• Multiple Detection Methods:

  • Validate findings using complementary techniques (e.g., mass spectrometry)

  • Compare results from both polyclonal and monoclonal antibodies

• Genetic Validation:

  • Perform knockdown/knockout experiments to confirm specificity

  • Use overexpression of tagged USP6 as a positive control

• Isoform Analysis:

  • Design primers for RT-PCR to identify which USP6 isoforms are expressed in your sample

  • Select antibodies that recognize conserved regions if multiple isoforms are present

What controls should be included to validate USP6 antibody specificity?

Comprehensive validation of USP6 antibody specificity requires multiple controls:

Positive Controls:

• Cell lines or tissues known to express USP6 (e.g., specific neoplasms with USP6 translocations)

• Recombinant USP6 protein (full-length or fragment corresponding to antibody epitope)

• Overexpression systems with tagged USP6 constructs

Negative Controls:

• USP6 knockout or knockdown samples (siRNA, shRNA, or CRISPR-Cas9)

• Cell lines known not to express USP6

• Competing peptide blocking experiments

Specificity Controls:

• Preimmune serum (for polyclonal antibodies)

• Isotype control antibodies (for monoclonal antibodies)

• Cross-reactivity assessment with related proteins (e.g., USP32)

Technical Controls:

• Secondary antibody-only controls to assess non-specific binding

• Multiple antibody dilutions to determine optimal signal-to-noise ratio

• Parallel testing with multiple antibody clones targeting different epitopes

How can researchers optimize USP6 antibody performance for challenging applications?

When standard protocols yield suboptimal results, consider these optimization strategies:

For Low Signal Intensity:

• Signal Amplification Methods:

  • Tyramide signal amplification (TSA) for immunohistochemistry/immunofluorescence

  • Enhanced chemiluminescence (ECL) substrates with higher sensitivity for Western blots

  • Consider using polyclonal antibodies that recognize multiple epitopes

• Sample Preparation Optimization:

  • For proteins resistant to extraction, test alternative lysis buffers

  • Extend antigen retrieval time for formalin-fixed tissues

  • Increase protein loading for Western blots (up to 80-100 μg)

For High Background:

• Protocol Adjustments:

  • Extend blocking time (2-3 hours or overnight)

  • Increase detergent concentration in washing buffers

  • Use alternative blocking agents (e.g., fish gelatin instead of BSA)

• Antibody Selection:

  • Switch to monoclonal antibodies for higher specificity

  • Consider directly conjugated primary antibodies to eliminate secondary antibody cross-reactivity

  • Test alternative clones targeting different epitopes

For Specialized Applications:

• Proximity-based Assays (PLA, FRET):

  • Use monoclonal antibodies with precisely characterized epitopes

  • Verify antibody compatibility with specialized reagents

• Fixed vs. Live Cell Imaging:

  • For live cell applications, test antibody fragments (Fab, scFv)

  • Optimize fixation protocols to preserve epitope accessibility

What approaches can help distinguish between USP6 and other closely related deubiquitinating enzymes?

Distinguishing USP6 from related proteins requires careful experimental design:

Comparative Expression Analysis:

• Employ multiple antibodies targeting different epitopes unique to USP6

• Compare with antibodies specific to related enzymes (e.g., USP32)

• Use species-specific differences (as USP6 is hominoid-specific)

Domain-Specific Detection:

• Target regions derived from the TBC1D3 portion of USP6, which is absent in USP32

• Focus on the chimeric junction region unique to USP6

• Utilize antibodies raised against specific functional domains

Functional Discrimination:

• Exploit USP6's unique ability to modulate ARF6 trafficking

• Leverage USP6's specific role in Wnt signaling via Frizzled deubiquitylation

• Assess deubiquitinase activity profiles with specific substrates

Advanced Analytical Techniques:

• Mass spectrometry following immunoprecipitation for definitive identification

• 2D gel electrophoresis to separate based on both molecular weight and isoelectric point

• RNA-seq analysis in parallel with protein detection to confirm identity

How are USP6 antibodies being utilized in cancer research and potential therapeutic development?

USP6 has been identified as oncogenic through chromosome translocation in certain human neoplasms . Advanced research applications include:

Diagnostic Applications:

• Immunohistochemical staining for USP6 to identify specific neoplasms with USP6 rearrangements

• Development of multiplexed detection systems combining USP6 antibodies with other diagnostic markers

• Liquid biopsy approaches detecting circulating USP6-expressing cells

Therapeutic Target Validation:

• Use of USP6 antibodies to monitor protein levels following treatment with potential inhibitors

• Correlation of USP6 expression/activity with treatment response

• Identification of critical interaction partners through co-immunoprecipitation studies

Mechanistic Studies:

• Investigation of USP6's role in Wnt signaling deregulation in cancer

• Analysis of matrix metalloproteinase production following USP6-mediated NF-κB activation

• Examination of deubiquitylation targets affecting cancer cell survival and proliferation

Translational Research Applications:

• Development of patient-derived xenograft models with validated USP6 expression

• High-throughput screening for compounds that modulate USP6 activity

• Correlation of USP6 expression/activity with clinical outcomes