USP17L6P Antibody, HRP 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 include cell proliferation, progression through the cell cycle, cell migration, and the cellular response to viral infection. Current research indicates that USP17L6P appears to be non-functional in the regulation of apoptosis, suggesting a specialized role in cellular homeostasis. As a member of the ubiquitin-specific processing protease family, it plays a critical role in protein turnover and cellular signaling pathways mediated by the ubiquitin-proteasome system.

What applications is the USP17L6P Antibody, HRP conjugated suitable for?

The USP17L6P Antibody, HRP conjugated has been specifically tested and validated for ELISA applications. The HRP (horseradish peroxidase) conjugation enables direct detection without the need for secondary antibodies, which can be particularly advantageous when working with complex experimental designs. While validated specifically for ELISA, researchers may potentially adapt this antibody for other applications where HRP-conjugated antibodies are commonly used, such as western blotting and immunohistochemistry, though additional validation would be required.

What is the significance of the HRP conjugation for detection methods?

The HRP conjugation provides several methodological advantages:

• Direct detection capability, eliminating the need for secondary antibodies

• Compatibility with multiple detection methods including:

  • Chemiluminescent detection (producing light emission)

  • Colorimetric detection (producing colored precipitates)

  • Fluorescent detection (with appropriate substrates)

HRP catalyzes the oxidation of substrates in the presence of hydrogen peroxide, resulting in either a colored precipitate or light emission, depending on the substrate used. This enzymatic amplification significantly enhances detection sensitivity compared to non-enzymatic labeling methods.

What buffer considerations are critical when working with USP17L6P Antibody, HRP conjugated?

The performance of HRP-conjugated antibodies is significantly influenced by buffer composition. For optimal results when working with USP17L6P Antibody, HRP conjugated, consider the following buffer parameters:

The antibody should be stored in conditions that avoid repeated freeze-thaw cycles to maintain conjugate integrity. Upon receipt, store at -20°C or -80°C. Working solutions should avoid components that can interfere with HRP activity or antibody binding, including sodium azide, which inhibits HRP activity.

How can I optimize an ELISA protocol using USP17L6P Antibody, HRP conjugated?

ELISA optimization requires systematic evaluation of multiple experimental variables. Based on experimental design techniques, researchers should consider:

• Critical primary factors:

  • Substrate incubation time (significant impact on signal development)

  • Enzyme label lot (source of variability between experiments)

  • Antibody dilution (requiring optimization for signal-to-noise ratio)

• Important interactions:

  • Dilutions of enzyme label and antibody show significant interaction effects and should be optimized together rather than independently

A methodical approach involves:

• Initial screening of 8-10 potential variables using fractional factorial design

• Follow-up factorial experiments focusing on the 3-4 most critical factors identified

• Evaluation using multiple performance criteria, including:

  • Standard curve reproducibility

  • Assay detection limits

  • Signal-to-background ratio

This systematic approach can reduce optimization time from years to months while providing robust, reproducible results.

What detection substrates are recommended for USP17L6P Antibody, HRP conjugated?

The choice of substrate depends on the desired detection method and sensitivity requirements:

• Colorimetric Detection:

  • Diaminobenzidine (DAB) + H₂O₂: Produces a brown water-insoluble precipitate

  • TMB (3,3',5,5'-tetramethylbenzidine): Produces a blue color that turns yellow when stopped with acid

  • ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)): Produces a green-blue soluble product

• Chemiluminescent Detection:

  • Enhanced chemiluminescence (ECL) substrates provide higher sensitivity for low-abundance targets

  • Luminol-based systems are most common and compatible with standard imaging equipment

• Fluorescent Detection:

  • Tyramide signal amplification systems can significantly enhance sensitivity when detecting low-abundance targets

Each substrate system offers different sensitivity ranges and stability characteristics. For quantitative applications, chemiluminescent substrates generally offer superior sensitivity and dynamic range.

How do I troubleshoot inconsistent results when using USP17L6P Antibody, HRP conjugated in ELISA?

Inconsistent results with HRP-conjugated antibodies can stem from multiple sources. A systematic troubleshooting approach should consider:

• Conjugate Stability Issues:

  • HRP conjugates naturally lose activity over time, especially with repeated freeze-thaw cycles

  • Performance diminishes with increasing temperature and dilution of conjugates

  • Consider using conjugate stabilizers like LifeXtend™ HRP conjugate stabilizer for working solutions

• Experimental Variables:

  • Substrate incubation time variability between experiments

  • Antibody dilution inconsistency

  • Temperature fluctuations during incubation steps

• Methodological Approach:

  • Implement standard curve quality control metrics in each assay

  • Use desirability functions to evaluate multiple response parameters simultaneously

  • Document lot numbers of critical reagents, as enzyme label lot can significantly impact results

• Technical Recommendations:

  • Prepare fresh working dilutions for each experiment

  • Maintain consistent timing between experiments

  • Consider using automated systems to reduce technical variability

How does the detection of USP17L6P compare to other deubiquitinating enzymes in cellular systems?

Detection of deubiquitinating enzymes (DUBs) like USP17L6P presents unique challenges compared to other protein families:

• Expression Dynamics:

  • USP17L6P expression is often tightly regulated and may be transiently expressed during specific cellular processes

  • Detection requires careful timing of sample collection to capture expression windows

• Specificity Considerations:

  • USP17L6P shares sequence similarity with other USP17 family members (USP17C, USP17D, USP17N)

  • Validate antibody specificity against recombinant proteins representing different family members

• Cellular Localization:

  • Consider subcellular fractionation protocols to enrich for compartments where USP17L6P is predominantly active

  • Whole cell lysates may dilute signal if expression is compartmentalized

• Activity vs. Expression:

  • Distinguish between detecting protein presence (expression) and enzyme activity (function)

  • Consider complementary activity-based probes when studying functional aspects

What are the critical considerations for using USP17L6P Antibody, HRP conjugated in studies of cell cycle regulation?

When investigating USP17L6P's role in cell cycle processes:

• Synchronization Protocols:

  • Cell populations should be synchronized to specific cell cycle phases

  • Consider double-thymidine block, serum starvation, or nocodazole treatment depending on the phase of interest

• Time-Course Experimental Design:

  • USP17L6P expression may fluctuate during cell cycle progression

  • Design sampling intervals to capture potential transient expression patterns

• Multiplexed Detection:

  • Co-stain for established cell cycle markers (cyclins, CDKs) to correlate USP17L6P expression with cell cycle phase

  • Consider sequential probing protocols to avoid cross-reactivity

• Quantification Methods:

  • Implement quantitative image analysis for immunocytochemistry

  • Use flow cytometry to correlate USP17L6P levels with DNA content

• Substrate Identification:

  • Consider co-immunoprecipitation approaches to identify potential substrates

  • Validate interactions with ubiquitination status analysis

What are the critical quality control parameters for USP17L6P Antibody, HRP conjugated?

Quality control for this antibody includes:

• Antibody Specificity:

  • Generated against recombinant Human Ubiquitin carboxyl-terminal hydrolase 17-like protein 6 protein (amino acids 136-398)

  • Polyclonal nature provides recognition of multiple epitopes

• Purification Method:

  • Protein G purification with >95% purity

  • IgG isotype confirmation

• Conjugation Quality:

  • HRP activity verification

  • Protein:enzyme ratio optimization

  • Functional testing in ELISA formats

• Storage Considerations:

  • Preservation in 50% glycerol with 0.03% Proclin 300 in PBS (pH 7.4)

  • Stability testing at recommended storage temperatures (-20°C or -80°C)

How can I validate cross-reactivity of USP17L6P Antibody, HRP conjugated with other USP17 family members?

The USP17 family contains several highly similar proteins. To validate specificity:

• Recombinant Protein Panel Testing:

  • Test against purified recombinant proteins of USP17C, USP17D, and USP17N

  • Quantify relative binding affinities using concentration-dependent ELISA

• Cellular Expression Systems:

  • Use cells with knockout/knockdown of specific USP17 family members

  • Compare signal intensity between wild-type and modified cells

• Peptide Competition Assays:

  • Pre-incubate antibody with peptides representing unique regions of different USP17 family members

  • Measure reduction in signal to identify epitope specificity

• Western Blot Migration Pattern:

  • Different USP17 family members may show subtle molecular weight differences

  • Compare migration patterns with predicted molecular weights

How can USP17L6P Antibody, HRP conjugated be applied to study viral infection responses?

USP17L6P is involved in cellular responses to viral infection. Research applications include:

• Temporal Expression Analysis:

  • Monitor USP17L6P expression at various time points post-infection

  • Correlate with viral replication kinetics

• Subcellular Redistribution:

  • Track potential changes in USP17L6P localization during infection

  • Co-stain with viral proteins to identify potential interactions

• Signaling Pathway Integration:

  • Examine USP17L6P in relation to innate immune signaling components

  • Focus on interferon regulatory pathways and NF-κB signaling

• Viral Antagonism:

  • Investigate whether specific viral proteins target USP17L6P function

  • Compare expression and activity across infections with different viruses

• Methodological Approach:

  • Implement time-course ELISA to quantify expression changes

  • Consider cell type-specific differences in USP17L6P regulation

What considerations are important when designing multiplexed detection systems including USP17L6P Antibody, HRP conjugated?

Multiplexed detection requires careful optimization:

• Compatible Detection Systems:

  • HRP can be combined with other enzyme systems (alkaline phosphatase, β-galactosidase)

  • Choose substrates with non-overlapping spectral properties

• Sequential Detection Protocols:

  • Consider order of detection to avoid masking low-abundance targets

  • Implement complete stripping protocols between detection cycles

• Cross-Reactivity Mitigation:

  • Validate antibody specificity in the presence of other detection reagents

  • Include appropriate blocking steps to minimize background

• Signal Separation Strategies:

  • Use mathematical deconvolution for overlapping signals

  • Implement reference standards for each target in the multiplex panel

• Technical Implementation:

  • Design plate layouts with appropriate controls for each detection system

  • Consider automated liquid handling to maintain consistent timing across steps

What emerging research areas might benefit from USP17L6P detection and functional characterization?

The role of USP17L6P in multiple cellular processes positions it as relevant to several emerging research domains:

• Cancer Biology:

  • Cell cycle dysregulation is a hallmark of cancer

  • USP17L6P's role in proliferation suggests potential involvement in oncogenic processes

• Immunotherapy Development:

  • Deubiquitinating enzymes are increasingly recognized as immunomodulators

  • USP17L6P may influence immune cell activation or exhaustion

• Viral Pathogenesis:

  • The involvement in viral infection responses suggests potential as a therapeutic target

  • May serve as a biomarker for specific viral infection stages

• Drug Discovery:

  • DUBs represent an emerging class of druggable targets

  • High-throughput screening assays utilizing this antibody could identify inhibitors

• Systems Biology:

  • Integration of USP17L6P into ubiquitin-proteasome system models

  • Network analysis of deubiquitinating enzyme coordination in cellular processes