USP10 Antibody, Biotin conjugated

What is USP10 and why is it significant in cellular research?

USP10 functions as a deubiquitylase (DUB) that removes ubiquitin from proteins, effectively preventing their degradation through proteasomal and lysosomal pathways . This enzyme plays a critical role in regulating the protein levels of integrin subunits β1 and β5, but interestingly not β3, demonstrating specificity in its targeting mechanism . The significance of USP10 extends to its involvement in promoting fibronectin recycling, with studies showing USP10 overexpression increases α5β1 integrin recycling by 1.9-fold and αv integrin recycling by 1.7-fold . Furthermore, USP10 has been identified as a regulator of p53 in DNA damage response and tumor development, positioning it as a crucial component in multiple cellular pathways . Understanding USP10 function provides insights into fundamental cellular processes including protein degradation, fibronectin matrix organization, and potentially cancer development mechanisms.

How do I select the appropriate USP10 antibody for my experimental needs?

Selection of an appropriate USP10 antibody should be guided primarily by your experimental application, target species, and the specific region of USP10 you aim to detect . For Western blotting applications, polyclonal antibodies like the rabbit polyclonal targeting AA 500-529 from the central region of human USP10 (ABIN1538461) or the polyclonal antibody 19374-1-AP can be effective choices, typically used at dilutions of 1:500-1:1000 . For immunofluorescence studies, antibodies validated for IF/ICC should be selected, with recommended dilution ranges typically around 1:50-1:500 . The reactivity profile is equally important—some USP10 antibodies react only with human samples, while others demonstrate cross-reactivity with mouse, rat, monkey, or dog samples . Consider also whether you need antibodies targeting specific regions of USP10, such as the N-terminal, C-terminal, or central regions, depending on your research question and the potential for interference from binding partners or post-translational modifications in your experimental system.

What are the validated applications for USP10 antibodies according to current research?

USP10 antibodies have been validated across multiple experimental applications according to current research literature and manufacturer specifications . Western blotting represents the most commonly verified application, with successful detection reported in cell lines including MCF-7 and HEK-293 . Immunoprecipitation applications have been validated using 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate, particularly effective in HEK-293 cells . Immunofluorescence and immunocytochemistry applications typically utilize dilutions of 1:50-1:500, with confirmed efficacy in HEK-293 cells . Additionally, USP10 antibodies have demonstrated utility in co-immunoprecipitation experiments for investigating protein-protein interactions, ELISA-based quantification, and immunohistochemistry applications for tissue samples . Research publications have also successfully employed these antibodies in knockdown/knockout validation studies, with at least 5 publications documenting this application .

Why might I consider using a biotin-conjugated USP10 antibody instead of unconjugated versions?

Biotin-conjugated USP10 antibodies offer significant advantages for certain experimental workflows due to the unique properties of the biotin-streptavidin interaction system. The extraordinary affinity between biotin and streptavidin (Kd ≈ 10^-15 M) provides one of the strongest non-covalent biological interactions, enabling highly sensitive detection systems. This conjugation eliminates the need for species-specific secondary antibodies, which is particularly advantageous when working with complex multi-labeling experiments where antibody cross-reactivity might present challenges . Biotin-conjugated antibodies can be readily detected using streptavidin coupled to various reporter molecules (fluorophores, enzymes, gold particles), providing flexibility across different detection platforms. Additionally, the biotin-streptavidin system offers signal amplification capabilities through the use of multiple biotin molecules per antibody, potentially enhancing detection sensitivity of low-abundance USP10 protein in samples where expression levels might be limited.

How can I effectively use USP10 antibodies to study integrin regulation pathways?

To effectively study integrin regulation pathways using USP10 antibodies, researchers should implement a multi-faceted experimental approach that combines protein level analysis with functional assessments . Begin with Western blotting to establish baseline expression levels of USP10 and integrin subunits (particularly αv, β1, and β5) in your model system, using validated antibodies at recommended dilutions (typically 1:500-1:1000) . For investigating the direct relationship between USP10 and integrin ubiquitination, implement ubiquitination assays combining USP10 knockdown (using validated siRNAs) with proteasomal inhibitors (MG132) and lysosomal inhibitors (chloroquine, bafilomycin A1), followed by immunoprecipitation of integrin subunits and detection of ubiquitin . Cell surface biotinylation assays provide crucial data on the impact of USP10 manipulation on integrin surface expression, as demonstrated in previous studies where USP10 overexpression increased surface levels of αv (1.9±0.4), β1 (3.0±1.2) and β5 (3.1±0.1) . For functional assessment, live cell confocal integrin recycling assays using fluorescently-labeled antibodies can quantify integrin trafficking dynamics, particularly relevant given that USP10 overexpression has been shown to increase α5β1 integrin recycling by 1.9-fold and αv integrin recycling by 1.7-fold .

What protocol should I follow to assess USP10's impact on protein ubiquitination using immunoprecipitation?

To assess USP10's impact on protein ubiquitination through immunoprecipitation, follow this detailed protocol based on established research methodologies . First, culture your cells of interest (such as human corneal fibroblasts or HEK-293 cells) and implement USP10 manipulation through either siRNA-mediated knockdown or plasmid-based overexpression . Prior to cell lysis, treat cells with both proteasomal inhibitors (10μM MG132 for 4-6 hours) and lysosomal inhibitors (100μM chloroquine or 100nM bafilomycin A1) to prevent degradation of ubiquitinated proteins . Lyse cells in RIPA buffer supplemented with 10mM EDTA (crucial for promoting the dissociation of integrin heterodimers), protease inhibitors, and deubiquitinase inhibitors (20mM N-ethylmaleimide) . For immunoprecipitation, incubate 500-1000μg of total protein with 2-4μg of antibody against your protein of interest (e.g., integrin β1 or β5) overnight at 4°C, followed by addition of protein A/G beads for 2-4 hours . After thorough washing with RIPA buffer, elute immunoprecipitated proteins by boiling in sample buffer containing DTT, separate by SDS-PAGE, and immunoblot using anti-ubiquitin antibodies . Alternatively, for in vitro deubiquitination assays, treat the immunoprecipitated proteins with recombinant active USP10 enzyme prior to SDS-PAGE to demonstrate direct deubiquitination activity .

How can I implement cell surface biotinylation assays to study USP10's effect on integrin trafficking?

Cell surface biotinylation assays provide crucial quantitative data on USP10's effect on integrin surface expression and trafficking dynamics . Begin by manipulating USP10 expression in your cellular model through either transient transfection with USP10 expression vectors (for overexpression studies) or USP10-targeting siRNAs (for knockdown studies) . For surface biotinylation, wash cells twice with ice-cold PBS (pH 8.0) and incubate with membrane-impermeable biotinylation reagent (typically sulfo-NHS-SS-biotin at 0.5mg/ml in PBS) for 30 minutes at 4°C, followed by quenching with 50mM Tris-HCl (pH 7.5) to stop the reaction . After cell lysis, incubate equal amounts of protein with streptavidin-agarose beads for 2 hours at 4°C to capture biotinylated (surface) proteins . Elute bound proteins by boiling in sample buffer containing DTT (which cleaves the disulfide bond in sulfo-NHS-SS-biotin), separate by SDS-PAGE, and immunoblot for integrins of interest (αv, β1, β5) . For recycling studies, first biotinylate surface proteins, allow internalization at 37°C, strip remaining surface biotin with a membrane-impermeable reducing agent (glutathione), then allow recycling at 37°C before stripping once more to measure the proportion of internalized integrins that recycled back to the surface .

What methods are recommended for visualizing USP10 and integrin colocalization using fluorescence microscopy?

For visualizing USP10 and integrin colocalization using fluorescence microscopy, implement a dual or triple immunolabeling approach with careful attention to antibody compatibility and optical setup . Culture cells on glass coverslips or glass-bottom dishes coated with appropriate extracellular matrix proteins (fibronectin at 10μg/ml is recommended for integrin studies) . For fixed-cell imaging, fix cells with 4% paraformaldehyde for 15 minutes, permeabilize with 0.1% Triton X-100 for 5 minutes, and block with 3% BSA in PBS for 1 hour . Apply primary antibodies against USP10 (dilution 1:50-1:500) and your integrin of interest in blocking buffer overnight at 4°C, followed by compatible fluorescently-labeled secondary antibodies . For improved resolution, consider super-resolution techniques such as structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy. For live-cell imaging of integrin trafficking, implement the previously validated live cell confocal integrin recycling assay: incubate cells with fluorescently-labeled anti-integrin antibodies at 4°C, allow internalization at 37°C, strip remaining surface antibodies with acid wash, then monitor recycling by live confocal microscopy . Quantify colocalization using established parameters such as Pearson's correlation coefficient or Manders' overlap coefficient through software packages like ImageJ with the JACoP plugin.

How can I design experiments to investigate USP10's role in TGFβ signaling pathways?

Designing experiments to investigate USP10's role in TGFβ signaling pathways requires a multi-pronged approach that links USP10's deubiquitinating activity to integrin-mediated TGFβ activation and downstream signaling events . Begin with establishing a cellular model system that demonstrates endogenous TGFβ signaling activity, such as the HEK-293t SMAD reporter cell line containing αv integrins that shows high endogenous TGFβ activity . Implement USP10 manipulation through either siRNA-mediated knockdown or plasmid-based overexpression, followed by assessment of TGFβ activity using luciferase-based SMAD reporter assays, which previous research has shown to decrease by 35±9% after USP10 silencing compared to control siRNA . To establish the connection between USP10, integrins, and TGFβ activation, conduct blocking experiments using function-blocking antibodies against αv integrins alongside USP10 overexpression, as previous research has demonstrated that blocking cell-surface αv integrins after USP10 overexpression reduced fibrotic markers . For downstream TGFβ signaling assessment, implement Western blotting for phosphorylated SMAD2/3, qRT-PCR for TGFβ-responsive genes (particularly fibrotic markers like α-SMA and fibronectin extra domain A), and immunofluorescence staining to visualize changes in protein localization and expression patterns in response to USP10 manipulation .

What are the considerations for implementing USP10 antibodies in flow cytometry experiments?

Implementing USP10 antibodies in flow cytometry experiments requires careful consideration of antibody selection, cell preparation, and experimental controls to ensure valid and reproducible results . Begin by selecting USP10 antibodies specifically validated for flow cytometry/FACS applications, such as the mouse monoclonal antibodies (clones 2E1, 1A10, or 1H9) that have been explicitly validated for FACS applications . Since USP10 is primarily an intracellular protein, cells must be fixed and permeabilized—use 4% paraformaldehyde for fixation (10 minutes at room temperature) followed by permeabilization with either 0.1% Triton X-100 (5 minutes) for total cellular USP10 or digitonin (50μg/ml for 10 minutes) for cytoplasmic-specific permeabilization . Titrate the primary USP10 antibody to determine optimal concentration, typically starting with manufacturer's recommendations and testing 2-fold dilutions above and below this concentration. For detection, use appropriate fluorophore-conjugated secondary antibodies or directly conjugated USP10 antibodies if available. Essential controls include isotype controls matching the primary antibody's host species and class, fluorescence-minus-one (FMO) controls, and positive biological controls such as cells with known USP10 overexpression or knockdown .

How can I develop a multiplex assay to simultaneously detect USP10 and its substrate proteins?

Developing a multiplex assay for simultaneous detection of USP10 and its substrate proteins requires careful selection of compatible detection systems and validation of potential cross-reactivity . For immunoblotting-based multiplex approaches, implement a multiplexed Western blot using primary antibodies from different host species (such as rabbit anti-USP10 and mouse anti-integrin antibodies) detected with species-specific secondary antibodies conjugated to distinct fluorophores on fluorescence-based imaging systems . For cellular imaging applications, design a multiplex immunofluorescence protocol utilizing primary antibodies from different host species against USP10 and its substrates (integrins αv, β1, β5, or SNX3), followed by species-specific secondary antibodies conjugated to spectrally distinct fluorophores . For high-throughput approaches, consider bead-based multiplex assays where antibodies against USP10 and its substrates are conjugated to differently coded beads, allowing simultaneous quantification in a single sample. To improve assay sensitivity for low-abundance proteins, incorporate signal amplification through tyramide signal amplification (TSA) or proximity ligation assay (PLA) techniques, particularly valuable for detecting transient USP10-substrate interactions . Validate your multiplex assay using positive controls (USP10 overexpression) and negative controls (USP10 knockdown) to confirm specificity and sensitivity across all detection channels .

How can I verify the specificity of my USP10 antibody in experimental applications?

Verifying the specificity of USP10 antibodies requires implementation of multiple complementary approaches to establish confidence in antibody performance . Begin with testing the antibody in a USP10 knockdown/knockout system—either siRNA-mediated knockdown or CRISPR/Cas9-mediated knockout—which should demonstrate significant reduction in signal intensity compared to control conditions . The literature documents at least 5 publications utilizing this approach for USP10 antibody validation . Perform Western blotting to confirm the antibody detects a protein of the expected molecular weight (100-130 kDa for USP10, which may vary due to post-translational modifications) in appropriate positive control samples such as MCF-7 or HEK-293 cells . For immunoprecipitation validation, conduct reciprocal co-immunoprecipitation experiments using multiple antibodies targeting different epitopes of USP10, followed by mass spectrometry analysis to confirm the presence of USP10-specific peptides in the immunoprecipitate . Consider epitope blocking experiments where pre-incubation of the antibody with its immunizing peptide (where available) should abolish or significantly reduce specific signal in your experimental system . For immunofluorescence applications, co-staining with multiple USP10 antibodies targeting different epitopes should demonstrate significant signal overlap in positive control samples.

What are the common pitfalls when using biotin-conjugated antibodies and how can I avoid them?

Using biotin-conjugated antibodies presents several common pitfalls that researchers should proactively address to ensure experimental success . Endogenous biotin interference represents a significant challenge, particularly in biotin-rich tissues like liver, kidney, and brain—pretreat samples with avidin/streptavidin blocking kits before applying biotin-conjugated antibodies to minimize this interference. Over-biotinylation of antibodies can lead to reduced antigen binding capacity due to modification of critical amino acids within the antigen-binding site—verify the degree of biotinylation and antibody functionality through comparison with unconjugated versions of the same antibody . Non-specific binding may occur through biotin-binding proteins in biological samples—implement thorough blocking steps using BSA (3-5%) or commercial biotin/avidin blocking systems before antibody application. When using multiple biotin-conjugated primary antibodies simultaneously, sequential detection with intermediate streptavidin blocking is necessary to prevent cross-reaction—apply the first biotin-conjugated antibody, detect with streptavidin-conjugate, block remaining biotin-binding sites with excess free biotin, then proceed with the second biotinylated antibody . For applications requiring signal amplification through multiple layers (e.g., biotinylated secondary antibody followed by streptavidin-HRP), optimize each amplification step separately to prevent excessive background signal.

What controls should I include when using USP10 antibodies in Western blotting experiments?

Comprehensive control implementation is essential for reliable Western blotting experiments using USP10 antibodies . Include a positive control sample with confirmed USP10 expression, such as MCF-7 or HEK-293 cells, which have been validated to express detectable levels of USP10 protein . Implement a negative control through USP10 knockdown or knockout samples—siRNA-mediated USP10 knockdown has been demonstrated to reduce USP10 protein levels by approximately 56±4% in previous studies . Include a loading control antibody targeting a housekeeping protein (GAPDH, β-actin, or α-tubulin) to normalize USP10 signal across samples with potential loading variations . For antibody specificity controls, consider peptide competition assays where pre-incubation of the antibody with its immunizing peptide should abolish specific binding . When comparing effects of treatments on USP10 expression, include appropriate vehicle controls for each treatment condition. For quantitative analyses, include a standard curve using recombinant USP10 protein at known concentrations, or implement a dilution series of a positive control sample to confirm signal linearity within your working range . For multiplex Western blotting, include single-antibody controls to verify lack of cross-reactivity between detection systems.

How can I optimize immunofluorescence protocols for detecting USP10 in different cell types?

Optimizing immunofluorescence protocols for USP10 detection across different cell types requires systematic adjustment of key parameters to maximize signal-to-noise ratio . Begin with fixation optimization—while 4% paraformaldehyde (15 minutes at room temperature) works well for most applications, methanol fixation (-20°C for 10 minutes) may better preserve certain epitopes; test both conditions to determine optimal preservation of your specific USP10 epitope . Permeabilization conditions significantly impact antibody accessibility to intracellular USP10—test a gradient of detergent concentrations (0.1-0.5% Triton X-100, 0.1-0.5% Saponin, or 50-100μg/ml Digitonin) and incubation times (5-15 minutes) to optimize for your specific cell type . Blocking conditions should be optimized based on the host species of your USP10 antibody—for rabbit polyclonal antibodies like 19374-1-AP, use 5% normal goat serum in PBS for 1 hour at room temperature . Antibody concentration requires careful titration, starting with the manufacturer's recommended dilution (1:50-1:500 for IF/ICC applications) and testing 2-fold dilutions above and below this range . For signal amplification in cells with low USP10 expression, implement tyramide signal amplification (TSA) or use high-sensitivity detection systems such as quantum dots. Finally, counterstain with DAPI for nuclear visualization and phalloidin for cytoskeletal context to better interpret USP10 localization patterns.

How might USP10 antibodies be utilized in studying neurodegenerative diseases?

USP10 antibodies present promising tools for investigating neurodegenerative disease mechanisms due to the emerging understanding of deubiquitination pathways in protein aggregation disorders . Begin by establishing USP10 expression profiles across different neural cell types (neurons, astrocytes, microglia) in both healthy and disease-state tissues using immunohistochemistry with validated USP10 antibodies . For Alzheimer's disease research, implement co-immunostaining of USP10 with amyloid-beta or tau to investigate potential associations, particularly given the role of ubiquitination in tau clearance mechanisms. In Parkinson's disease models, examine USP10 localization relative to α-synuclein aggregates using high-resolution microscopy techniques such as structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy . Functional studies should incorporate USP10 manipulation (overexpression or knockdown) in neural cell cultures followed by assessment of protein aggregation, cell viability, and autophagy markers . Extend investigations to animal models of neurodegeneration using USP10 antibodies for immunohistochemical analysis of brain sections, potentially revealing alterations in USP10 expression or localization during disease progression . For mechanistic insights, combine USP10 antibodies with antibodies against specific ubiquitin chain linkages (K48, K63) to determine the types of ubiquitin modifications being regulated by USP10 in neural contexts.

What are the considerations for incorporating USP10 antibodies in tissue microarray (TMA) analysis?

Incorporating USP10 antibodies in tissue microarray (TMA) analysis requires careful optimization of immunohistochemistry protocols and comprehensive validation strategies . Begin with antibody selection, prioritizing USP10 antibodies validated specifically for immunohistochemistry applications, such as those documented to work in paraffin-embedded tissues . Optimization of antigen retrieval methods is critical—test both heat-induced epitope retrieval (citrate buffer pH 6.0 and EDTA buffer pH 9.0) and enzymatic retrieval (proteinase K) to determine which best exposes the USP10 epitope while preserving tissue morphology . For detection systems, compare chromogenic (DAB) versus fluorescent approaches, considering that fluorescence may offer advantages for multiplex analysis but may be more susceptible to autofluorescence in certain tissues . Establish and validate a quantitative scoring system for USP10 expression—consider both staining intensity (0-3+) and percentage of positive cells to generate H-scores or Allred scores for systematic comparison across tissues . Include appropriate controls in your TMA design: positive control tissues with known USP10 expression, negative control tissues, and technical controls (omitting primary antibody) . For validation, perform parallel Western blotting analysis on matched tissue lysates to confirm the specificity of the USP10 antibody in your tissue of interest . Consider digital pathology approaches for automated, objective quantification of USP10 staining across large TMA cohorts.

How can USP10 antibodies contribute to research on fibronectin-dependent tissue fibrosis?

USP10 antibodies can significantly advance research on fibronectin-dependent tissue fibrosis through multiple experimental approaches targeting the USP10-integrin-fibronectin axis . Implement immunohistochemistry in fibrotic tissue samples using USP10 antibodies alongside markers for activated fibroblasts (α-SMA) and fibronectin deposition (especially the FN-EDA splice variant associated with fibrosis) to establish correlations between USP10 expression and fibrotic progression . In cellular models of fibrosis, utilize USP10 antibodies for Western blotting and immunofluorescence to track changes in USP10 expression during myofibroblast activation and persistence . Functional studies should incorporate manipulation of USP10 levels (overexpression/knockdown) followed by assessment of fibronectin matrix organization—previous research has demonstrated that USP10 overexpression increased biotinylated fibronectin internalization (2.1-fold) and recycling over 4 days (1.7–2.2-fold) . For mechanistic investigations, implement co-immunoprecipitation using USP10 antibodies to identify novel binding partners in fibroblasts during fibrotic activation . Develop ex vivo fibrosis models where tissue explants are cultured with USP10 inhibitors or activators, followed by immunohistochemical analysis of fibronectin organization and collagen deposition. In vivo studies in fibrosis models should incorporate USP10 antibodies for tissue analysis, potentially revealing therapeutic targets within the USP10-integrin-fibronectin pathway.

What potential exists for using USP10 antibodies in developing targeted therapeutic approaches?

USP10 antibodies hold significant potential for developing targeted therapeutic approaches through both diagnostic and mechanistic applications . For diagnostic applications, USP10 antibodies could enable identification of patient populations with altered USP10 expression patterns that might benefit from targeted therapies—implement immunohistochemistry or flow cytometry using validated USP10 antibodies to stratify patients in clinical studies . As research tools, USP10 antibodies can facilitate high-throughput screening of USP10 inhibitors by establishing cellular assays measuring USP10 protein levels, subcellular localization, or post-translational modifications in response to candidate compounds . For potential direct therapeutic applications, consider developing function-blocking antibodies targeting USP10, which could attenuate pathological deubiquitination activity in conditions where USP10 is overactive, such as fibrotic diseases where USP10 promotes excessive integrin accumulation and TGFβ signaling . USP10-targeting antibody-drug conjugates (ADCs) could specifically deliver cytotoxic payloads to cells overexpressing USP10, representing another potential therapeutic strategy. For developing biomarker applications, validate whether USP10 protein levels (detected using specific antibodies) correlate with disease progression or therapeutic response in relevant pathological conditions . Finally, USP10 antibodies can facilitate fundamental research on USP10 structure-function relationships, potentially informing structure-based drug design of small molecule USP10 inhibitors.

What are the comparative specifications of available USP10 antibodies for research applications?

USP10 antibodies currently available for research display diverse properties that should guide selection based on specific experimental requirements. The following table summarizes key specifications across commonly used USP10 antibodies:

When selecting a USP10 antibody, consider the balance between polyclonal antibodies (offering broader epitope recognition) versus monoclonal antibodies (providing greater consistency between lots) . For biotin conjugation projects, antibodies with demonstrated high affinity and specificity in their unconjugated form should be prioritized to ensure functionality is maintained after conjugation .

How does USP10 manipulation affect integrin and fibronectin dynamics in experimental systems?

The complex relationship between USP10 manipulation and integrin/fibronectin dynamics has been quantitatively documented across several experimental systems. The following table summarizes key findings:

These quantitative data demonstrate that USP10 exhibits specificity in its deubiquitylating activity, preferentially targeting β1 and β5 integrin subunits but not β3 . This specificity translates into differential regulation of integrin protein levels, with USP10 manipulation having the most pronounced effect on β1 integrins (3.9±1.1-fold increase with overexpression) . Importantly, these changes in integrin levels directly impact downstream cellular processes, including fibronectin recycling and TGFβ signaling, highlighting the potential significance of targeting USP10 in fibrotic diseases .