USP17L6P Antibody, Biotin conjugated

What is USP17L6P and what cellular processes does it regulate?

USP17L6P (Ubiquitin carboxyl-terminal hydrolase 17-like protein 6) is a deubiquitinating enzyme that removes conjugated ubiquitin from specific proteins to regulate various cellular processes. These processes include cell proliferation, progression through the cell cycle, cell migration, and cellular response to viral infection. Current research indicates that USP17L6P appears to be non-functional in the regulation of apoptosis . The enzyme is part of the broader USP (Ubiquitin-Specific Protease) family, which plays crucial roles in protein homeostasis by reversing ubiquitination and thereby preventing protein degradation through the ubiquitin-proteasome pathway.

Why is a biotin conjugation selected for USP17L6P antibodies, and what advantages does this provide?

Biotin conjugation is selected for USP17L6P antibodies because it leverages the exceptionally high-affinity interaction between biotin and streptavidin (dissociation constant Kd ≈ 10⁻¹⁵ M), creating one of the strongest non-covalent biological bonds . This conjugation offers several significant advantages for research applications:

• Signal amplification: The tetravalent binding of streptavidin to biotin enables multiple layers of detection, enhancing sensitivity for low-abundance targets like USP17L6P .

• Versatility across detection methods: Biotin-conjugated antibodies can be visualized using various streptavidin conjugates (HRP, fluorophores, gold particles), making them adaptable for multiple experimental platforms .

• Improved spatial resolution: The small size of biotin (244 Da) minimizes steric hindrance between the antibody and detection system, improving access to antigens in complex tissue structures .

• Multi-layered detection systems: The biotin-streptavidin system allows for building multi-step detection protocols that can significantly enhance signal-to-noise ratios .

How does USP17L6P function at the molecular level and what are its key structural features?

USP17L6P functions as a cysteine protease that hydrolyzes the isopeptide bond between ubiquitin and target proteins. While the exact crystal structure of USP17L6P has not been fully characterized in the provided search results, its function can be understood through its homology to other USP family members:

• Catalytic mechanism: USP17L6P contains a catalytic triad (likely including a cysteine residue) typical of deubiquitinating enzymes that cleaves the isopeptide bond between ubiquitin and substrate proteins .

• Substrate recognition: The enzyme likely contains substrate-binding domains that determine its specificity for particular ubiquitinated proteins involved in cell cycle regulation and migration.

• Regulatory domains: As with other USPs, USP17L6P likely contains regulatory domains that modulate its catalytic activity in response to cellular conditions.

The protein sequence (particularly amino acids 136-398) appears to contain immunogenic epitopes that are used as targets for antibody production .

What are the optimal protocols for using USP17L6P antibody, biotin conjugated in ELISA applications?

For optimal ELISA performance with biotin-conjugated USP17L6P antibody, the following methodological approach is recommended:

Standard ELISA Protocol:

• Plate Coating: Coat microplate wells with capture antibody (anti-USP17L6P) at 1-10 μg/mL in coating buffer (typically carbonate-bicarbonate buffer pH 9.6) overnight at 4°C.

• Blocking: Block non-specific binding sites with 1-5% BSA or casein in PBS for 1-2 hours at room temperature.

• Sample Addition: Add samples containing USP17L6P protein and incubate for 1-2 hours at room temperature.

• Detection Antibody: Add biotin-conjugated USP17L6P antibody (typically at 1:500 dilution based on optimization experiments) and incubate for 1 hour at room temperature .

• Streptavidin-HRP Addition: Add streptavidin-HRP conjugate (1:1000 to 1:5000 dilution) and incubate for 30-45 minutes.

• Substrate Addition: Add appropriate substrate (TMB for HRP) and monitor color development.

• Stopping Reaction: Add stop solution (typically 2N H₂SO₄ for TMB) when adequate color develops.

• Reading: Measure absorbance at appropriate wavelength (450 nm for TMB).

What controls and validation steps should be included when using biotin-conjugated USP17L6P antibody?

A comprehensive validation strategy for biotin-conjugated USP17L6P antibody experiments should include:

Essential Controls:

• Positive Control: Recombinant USP17L6P protein (preferably the 136-398AA region used as immunogen) to confirm antibody functionality.

• Negative Controls:

  • Isotype control: Biotin-conjugated rabbit IgG with no specific target

  • Substrate omission: Complete protocol without primary antibody

  • Known negative samples: Tissues/cells with confirmed absence of USP17L6P expression

Validation Steps:

• Specificity Testing:

  • Western blot analysis showing a single band at the expected molecular weight (~60 kDa)

  • Cross-reactivity assessment with related USP family members (USP17C, USP17D, USP17N)

• Sensitivity Determination:

  • Serial dilution of positive control to establish detection limits (typically in the 250 pM range for biotin-streptavidin systems)

  • Comparison with non-biotinylated antibody detection systems

• Reproducibility Assessment:

  • Technical replicates (minimum triplicate)

  • Inter-assay variability measurement

• Endogenous Biotin Blocking:

  • Include avidin/biotin blocking steps when working with tissues known to have high endogenous biotin (e.g., liver, kidney)

What are the storage and handling recommendations to maintain optimal activity of biotin-conjugated USP17L6P antibody?

To preserve the functionality of biotin-conjugated USP17L6P antibody, adhere to these evidence-based storage and handling guidelines:

Storage Conditions:

• Short-term (up to 1 month): Store at 4°C with preservative (typically 0.03% Proclin 300) .

• Long-term: Store at -20°C or -80°C, preferably in small working aliquots to avoid repeated freeze-thaw cycles .

• Buffer System: Optimal storage buffer typically contains 50% glycerol, 0.01M PBS (pH 7.4), and preservative .

Handling Recommendations:

• Avoid repeated freeze-thaw cycles, which can lead to biotin conjugate degradation and reduced binding affinity.

• Centrifuge the product briefly before opening if not completely clear after storage.

• Dilute only immediately before use to maintain stability.

• For reconstitution of lyophilized antibody, use deionized water or recommended buffer.

• After reconstitution, store working aliquots at recommended temperatures to prevent degradation.

Stability Parameters:

• Typical shelf life: 12 months from date of receipt when stored properly .

• Activity testing: Periodically verify antibody activity using a small aliquot in a controlled ELISA.

• Visible indicators of degradation: Cloudiness, precipitation, or significantly reduced signal compared to fresh antibody.

How can non-specific binding be minimized when using biotin-conjugated USP17L6P antibody in tissue sections?

Non-specific binding is a common challenge when using biotin-conjugated antibodies in tissue sections. To minimize this issue with USP17L6P antibody, implement the following methodological approaches:

Endogenous Biotin Blocking:

• Prior to primary antibody incubation, block endogenous biotin using an avidin/biotin blocking kit .

• Apply avidin solution (0.1-1 mg/mL) for 15 minutes, wash, then apply biotin solution (0.1-1 mg/mL) for another 15 minutes.

Optimization of Blocking Reagents:

• Use a combination of 2-5% BSA, 5-10% normal serum (from the same species as the secondary antibody), and 0.1-0.3% Triton X-100 or Tween-20 in PBS.

• For tissues with high background, add 0.1-1% non-fat dry milk to the blocking solution.

• Consider specialty blocking reagents for tissues with high endogenous biotin (e.g., liver, kidney, brain).

Pre-adsorption Strategies:

• Use highly cross-adsorbed secondary antibodies to reduce cross-reactivity with endogenous immunoglobulins .

• Pre-adsorb the biotin-conjugated antibody with tissue powder from the species being tested.

Optimization Table for Reducing Non-specific Binding:

What strategies can improve signal-to-noise ratio when detecting low abundance USP17L6P in complex samples?

Detecting low-abundance USP17L6P in complex samples requires specialized approaches to enhance signal while minimizing background noise:

Signal Amplification Strategies:

Background Reduction Methods:

• Sample Pre-clearing:

  • Pre-incubate samples with non-immune serum and protein A/G beads

  • Remove high-abundance proteins using appropriate depletion methods

• Optimized Washing:

  • Increase washing stringency with higher salt concentrations (up to 300 mM NaCl)

  • Add 0.05-0.1% Tween-20 to wash buffers

  • Implement longer and more frequent washing steps

• Cross-reactivity Elimination:

  • Use highly cross-adsorbed secondary antibodies

  • Pre-adsorb antibodies with proteins from non-target species

Optimization Table for Signal-to-Noise Enhancement:

How can biotinylated USP17L6P antibody performance be assessed when experiments yield inconsistent results?

When experiments with biotinylated USP17L6P antibody produce inconsistent results, a systematic troubleshooting approach is necessary:

Antibody Validation Assessment:

• Activity Testing:

  • Perform dot blot with positive control (recombinant USP17L6P protein)

  • Compare signal intensity to a reference standard or previous lot

  • Quantify biotin conjugation level using HABA assay (4'-hydroxyazobenzene-2-carboxylic acid)

• Specificity Confirmation:

  • Western blot analysis with positive and negative control samples

  • Competitive binding assay with unconjugated antibody

  • Peptide blocking with immunogenic peptide (amino acids 136-398 of USP17L6P)

Methodological Troubleshooting Matrix:

Statistical Quality Control:

• Calculate intra-assay coefficient of variation (CV) from technical replicates (acceptable: <10%)

• Calculate inter-assay CV from multiple experiments (acceptable: <15%)

• Implement Levey-Jennings charts to track assay performance over time

• Use Westgard rules to identify systematic errors in the assay

If inconsistency persists despite these measures, consider:

• Testing antibodies from multiple suppliers or lots

• Switching to alternative detection methods (e.g., direct HRP conjugation)

• Verifying target protein expression through independent methods (e.g., RT-PCR)

How can biotin-conjugated USP17L6P antibody be effectively used in multiplex immunoassays?

Multiplex immunoassays allow simultaneous detection of multiple targets including USP17L6P, offering advantages in sample conservation, internal controls, and pathway analysis. Here's a methodological approach for incorporating biotin-conjugated USP17L6P antibody in multiplex systems:

Bead-Based Multiplex Strategy:

• Antibody Coupling to Distinct Bead Sets:

  • Conjugate capture antibodies for USP17L6P and other targets to spectrally distinct beads

  • Ensure adequate separation of emission spectra to prevent signal overlap

  • Validate each antibody pair independently before multiplexing

• Optimization for USP17L6P Detection:

  • Determine optimal biotin-conjugated USP17L6P antibody concentration through titration (typically 0.5-5 μg/mL)

  • Test for cross-reactivity with other detection antibodies in the multiplex panel

  • Address potential interference through strategic selection of detection fluorophores

• Signal Discrimination Strategy:

  • Utilize streptavidin conjugated to a unique fluorophore (e.g., PE, APC) with minimal spectral overlap

  • Apply appropriate compensation matrices to correct for spectral overlap

  • Consider tandem dyes for improved separation in complex panels

Planar Multiplex Approach:

• Spatial Separation Method:

  • Print capture antibodies in defined locations on planar surfaces

  • Apply sample and detect with a cocktail of detection antibodies, including biotin-conjugated USP17L6P antibody

  • Visualize using spectrally distinct streptavidin conjugates or tyramide signal amplification

• Sequential Detection Protocol:

  • For targets requiring different detection conditions, implement sequential detection with stripping between rounds

  • Optimize stripping conditions to remove previous detection antibodies without affecting immobilized proteins

  • Carefully validate signal persistence after each stripping cycle

Technical Considerations for Multiplexing with Biotin-Conjugated USP17L6P Antibody:

What advanced techniques can be used to study USP17L6P protein-protein interactions using biotin-conjugated antibodies?

Biotin-conjugated USP17L6P antibodies can be leveraged in sophisticated protein-protein interaction studies using the following methodological approaches:

Proximity Ligation Assay (PLA):

• Basic Protocol:

  • Target USP17L6P with biotin-conjugated antibody and potential interaction partner with a different primary antibody

  • Add PLA probes (streptavidin-oligonucleotide and secondary antibody-oligonucleotide)

  • Perform ligation when probes are in proximity (<40 nm)

  • Amplify DNA circle via rolling circle amplification

  • Detect with fluorescent probes

• Optimization for USP17L6P Studies:

  • Carefully titrate biotin-conjugated USP17L6P antibody concentration

  • Test multiple antibody combinations targeting different epitopes

  • Include appropriate controls to validate interaction specificity

Biotin-Based Pull-Down Coupled with Mass Spectrometry:

• Methodology:

  • Immunoprecipitate USP17L6P complexes using biotin-conjugated antibody

  • Capture using streptavidin magnetic beads

  • Elute under native or denaturing conditions based on downstream applications

  • Analyze interaction partners via liquid chromatography-tandem mass spectrometry (LC-MS/MS)

• Enhanced Stringency Protocol:

  • Implement cross-linking prior to lysis to capture transient interactions

  • Use stepped elution protocols to discriminate between high and low-affinity interactions

  • Apply quantitative approaches (SILAC, TMT) to distinguish specific from non-specific binding

FRET-Based Interaction Analysis:

• Implementation:

  • Detect USP17L6P with biotin-conjugated antibody, followed by streptavidin-conjugated donor fluorophore

  • Target potential interaction partner with acceptor fluorophore-conjugated antibody

  • Measure energy transfer as indicator of protein proximity

• Quantitative Assessment:

  • Calculate FRET efficiency to estimate interaction distance

  • Perform acceptor photobleaching to confirm FRET signal validity

  • Use time-resolved FRET for improved signal-to-noise ratio

Data Analysis and Validation Strategy:

These advanced approaches can reveal USP17L6P's interaction network, potentially identifying novel substrates regulated through deubiquitination and providing insights into its role in cellular processes including cell cycle regulation and migration.

How can researchers differentiate between the roles of USP17L6P and other USP family members using biotin-conjugated antibodies?

Distinguishing the specific functions of USP17L6P from other highly homologous USP family members requires sophisticated experimental approaches using biotin-conjugated antibodies:

Selective Depletion Strategy:

• Immunodepletion Protocol:

  • Conjugate biotin-USP17L6P antibody to streptavidin-coated magnetic beads

  • Deplete USP17L6P from cellular lysates while leaving other USP members

  • Compare enzyme activity profiles of depleted versus non-depleted samples

  • Rescue experiments with recombinant USP17L6P to confirm specificity

• Sequential Immunodepletion:

  • Perform ordered immunodepletion of multiple USP family members

  • Analyze residual deubiquitinating activity after each depletion step

  • Quantify the contribution of each family member to total activity

Substrate Specificity Assessment:

• Ubiquitin Chain-Specific Analysis:

  • Prepare substrate panels with different ubiquitin linkages (K48, K63, linear, etc.)

  • Immunoprecipitate USP17L6P using biotin-conjugated antibody

  • Assess activity against different ubiquitin chain topologies

  • Compare with activity profiles of other immunoprecipitated USP family members

• Comparative Substrate Profiling:

  • Identify potential substrates through proteomic approaches

  • Validate using biotin-conjugated antibodies for multiple USP family members

  • Determine substrate preference through competitive binding assays

Functional Genomics Approach:

• Combined Knockdown and Overexpression:

  • Systematically deplete individual USP family members

  • Rescue with biotin-tagged USP17L6P or other family members

  • Track cellular phenotypes to determine functional redundancy

• Domain Swapping Analysis:

  • Create chimeric proteins between USP17L6P and related USPs

  • Express biotin-tagged chimeras in cellular models

  • Determine which domains confer functional specificity

Comparative Analysis of USP Family Members:

This systematic approach allows researchers to delineate the unique roles of USP17L6P compared to closely related family members, which is crucial for understanding its specific contributions to cellular physiology and potential as a therapeutic target.

What emerging technologies might enhance the utility of biotin-conjugated USP17L6P antibody for single-cell protein analysis?

Recent advances in single-cell technologies present exciting opportunities for using biotin-conjugated USP17L6P antibodies to study cell-to-cell variability in deubiquitinating enzyme activity. The following methodological approaches represent cutting-edge applications:

Mass Cytometry (CyTOF) Applications:

• Metal-Tagged Streptavidin Strategy:

  • Detect biotin-conjugated USP17L6P antibody with lanthanide-labeled streptavidin

  • Simultaneously measure multiple proteins (30+ parameters) at single-cell resolution

  • Correlate USP17L6P expression with cell cycle markers, signaling molecules, and potential substrates

  • Implement algorithms like SPADE or viSNE for high-dimensional data visualization

• Multiplexed Tissue Imaging:

  • Apply Imaging Mass Cytometry (IMC) or Multiplexed Ion Beam Imaging (MIBI) with biotin-conjugated USP17L6P antibody

  • Visualize spatial distribution at subcellular resolution

  • Correlate with tissue architecture and microenvironment features

Proximity-Based Single-Cell Protein Analysis:

• Single-Cell Proximity Extension Assay (PEA):

  • Combine biotin-conjugated USP17L6P antibody with DNA-conjugated detection antibody

  • Generate amplifiable DNA templates when antibodies bind in proximity

  • Quantify at single-cell level through microfluidic platforms

  • Profile USP17L6P simultaneously with 40+ other proteins

Microfluidic Approaches:

• Single-Cell Western Blotting:

  • Separate proteins from individual cells in microfluidic chambers

  • Detect USP17L6P using biotin-conjugated antibody and fluorescent streptavidin

  • Correlate with other proteins from the same individual cell

  • Quantify expression level heterogeneity across cell populations

• Droplet-Based Enzymatic Assays:

  • Encapsulate single cells with biotin-conjugated USP17L6P antibody and fluorogenic deubiquitinating substrates

  • Measure enzymatic activity at single-cell resolution

  • Sort cells based on activity levels for downstream analysis

Technical Innovation Opportunities:

These emerging technologies will enable researchers to address fundamental questions about USP17L6P's role in cellular heterogeneity, its dynamic regulation during cell cycle progression, and its differential activity in response to various cellular stressors at unprecedented resolution.

How might researchers optimize biotin-conjugated USP17L6P antibodies for therapeutic target validation studies?

As USP17L6P's potential as a therapeutic target emerges, especially in cancer and viral infection contexts, biotin-conjugated antibodies can play crucial roles in target validation. The following methodological approaches outline optimization strategies:

Target Engagement Assessment:

• Cellular Thermal Shift Assay (CETSA):

  • Treat cells with potential USP17L6P inhibitors

  • Heat-challenge to denature unbound protein

  • Immunoprecipitate with biotin-conjugated USP17L6P antibody

  • Quantify thermal stability shifts as indicator of compound binding

• Biolayer Interferometry (BLI) Approach:

  • Immobilize biotin-conjugated USP17L6P antibody on streptavidin biosensors

  • Capture native USP17L6P from cell lysates

  • Measure compound binding kinetics in real-time

  • Determine residence time and binding affinity parameters

Pathway-Specific Functional Validation:

• Substrate-Specific Deubiquitination Assays:

  • Identify physiological substrates using biotin-conjugated USP17L6P antibody pull-downs

  • Develop substrate-specific activity assays

  • Test inhibitor effects on specific substrate deubiquitination

  • Correlate with cellular phenotypes

• Spatiotemporal Activity Profiling:

  • Use biotin-conjugated activity-based probes (ABPs) that bind active USP17L6P

  • Visualize active enzyme localization with fluorescent streptavidin

  • Monitor inhibitor effects on enzyme activity and localization

  • Correlate with cell cycle phases and stress responses

In Vivo Target Validation:

• Antibody-Drug Conjugate (ADC) Approach:

  • Utilize biotin-conjugated USP17L6P antibody to deliver compounds to target cells

  • Assess target engagement in complex tissues

  • Evaluate pharmacodynamic biomarkers in animal models

  • Correlate with efficacy and toxicity profiles

Comparative Assessment Framework:

This systematic approach not only validates USP17L6P as a therapeutic target but also provides critical insights into compound mode of action, potential resistance mechanisms, and biomarkers for clinical development.

What computational approaches can enhance analysis of data generated using biotin-conjugated USP17L6P antibodies?

Modern computational methods can significantly enhance the extraction of biological insights from experiments using biotin-conjugated USP17L6P antibodies. The following methodological approaches represent current best practices:

Network Analysis for Interaction Data:

• Protein-Protein Interaction (PPI) Network Construction:

  • Generate interaction data using biotin-conjugated USP17L6P antibody pull-downs

  • Apply unsupervised clustering algorithms to identify functional modules

  • Implement weighted correlation network analysis to find co-regulated partners

  • Integrate with public PPI databases to contextualize novel interactions

• Pathway Enrichment Analysis:

  • Map USP17L6P interactors to canonical pathways

  • Implement Gene Set Enrichment Analysis (GSEA) for interactome data

  • Apply topology-based enrichment methods that consider network structure

  • Visualize with tools like Cytoscape with EnrichmentMap plugins

Machine Learning Applications:

• Predictive Modeling of USP17L6P Substrates:

  • Train algorithms on known deubiquitinating enzyme substrates

  • Extract sequence and structural features from pull-down data

  • Apply supervised classification methods to predict novel substrates

  • Validate high-confidence predictions experimentally

• Image Analysis Automation:

  • Develop deep learning algorithms for pattern recognition in immunofluorescence using biotin-conjugated USP17L6P antibodies

  • Implement instance segmentation for single-cell analysis

  • Quantify subcellular localization changes under various conditions

  • Correlate spatial patterns with functional outcomes

Integrative Multi-Omics Analysis:

• Data Integration Framework:

  • Combine USP17L6P proteomics data with transcriptomics and ubiquitinome profiling

  • Implement multi-block data fusion techniques (MOFA, DIABLO)

  • Apply tensor factorization for time-series multi-omics data

  • Identify context-specific regulation patterns

• Causality Inference:

  • Construct Bayesian networks from perturbation experiments

  • Infer directionality in USP17L6P-centered regulatory networks

  • Apply dynamic causal modeling for time-resolved data

  • Identify key intervention points in regulatory cascades

Computational Analysis Workflow Comparison: