USP17L6P Antibody, FITC conjugated

What is USP17L6P Antibody and what are its key properties?

USP17L6P Antibody recognizes Ubiquitin carboxyl-terminal hydrolase 17-like protein 6 (EC 3.4.19.12), also known as Deubiquitinating enzyme 17-like protein 6, Ubiquitin thioesterase 17-like protein 6, or Ubiquitin-specific-processing protease 17-like protein 6. The FITC-conjugated version (Product Code: CSB-PA757690LC01HU) is a rabbit polyclonal antibody (IgG isotype) that has been conjugated to fluorescein isothiocyanate (FITC) for direct fluorescence detection. The antibody was raised against recombinant Human Ubiquitin carboxyl-terminal hydrolase 17-like protein 6 protein (136-398AA) and demonstrates reactivity specifically with human samples. It is provided in liquid form, purified using Protein G (>95% purity) .

What are the optimal storage conditions for FITC-conjugated antibodies?

FITC-conjugated antibodies should be stored at 4°C protected from exposure to light upon initial receipt. For long-term storage, it is recommended to aliquot the antibody and store at -20°C or -80°C, also protected from light exposure. Continuous exposure to light will gradually reduce the fluorescence intensity of FITC conjugates. The storage buffer for USP17L6P Antibody, FITC conjugated contains 0.03% Proclin 300 as a preservative, with 50% Glycerol and 0.01M PBS at pH 7.4 as constituents .

Repeated freeze-thaw cycles should be strictly avoided as they can significantly reduce antibody activity. When properly stored, the product is guaranteed for six months from the date of receipt .

How should I prepare working dilutions of FITC-conjugated antibodies for experiments?

When preparing working dilutions of FITC-conjugated antibodies:

• Always thaw frozen aliquots completely at room temperature before use

• Mix gently by pipetting or flicking the tube (avoid vortexing)

• For immunofluorescence applications on mammalian cells, a 1:500 dilution in Phosphate-Buffered Saline (PBS) containing 10% fetal bovine serum (FBS) is typically recommended as a starting point

• Prepare working solutions immediately before use and keep protected from light

• Centrifuge briefly before opening the vial to ensure all liquid is at the bottom

It's important to note that optimal dilutions may vary depending on your specific application, sample type, and cell line. Therefore, empirical determination of appropriate antibody concentration may be necessary for your particular experimental conditions .

What are the validated applications for USP17L6P Antibody, FITC conjugated in research?

While specific application data for USP17L6P Antibody is limited in the provided information, FITC-conjugated antibodies are generally suitable for several research applications including:

• Immunofluorescence microscopy - For direct visualization of target proteins in fixed cells or tissues

• Flow cytometry - For quantitative analysis of target protein expression in cell populations

• Western blot analysis - Can be used with appropriate secondary detection systems

• ELISA and Dot Blot assays - For quantitative detection of target proteins

For certain FITC-conjugated antibodies, specific applications have been validated, such as ELISA and Dot Blot for the FITC-conjugated uspF antibody . When designing experiments with USP17L6P Antibody, FITC conjugated, researchers should validate the antibody for their specific application and experimental system.

What is the detailed protocol for immunofluorescence detection using FITC-conjugated antibodies?

The following immunofluorescence protocol for cultured cells is recommended for FITC-conjugated antibodies:

Materials Required:

• Phosphate-Buffered Saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄·7H₂O, 1.4 mM KH₂PO₄, pH 7.3)

• Fetal Bovine Serum (FBS)

• Methanol (or alternative fixative)

• Blocking buffer (PBS + 10% FBS)

• FITC-conjugated antibody

• Fluorescence microscope with appropriate FITC filter

Procedure:

• Plate cells in a 35-mm dish or a single well in a six-well tissue culture plate and incubate overnight at 37°C until approximately 50% confluence is reached

• If expressing recombinant proteins, transfect cells and allow appropriate time for protein expression

• Remove culture medium and wash cells twice with PBS

• Fix cells by adding 2 mL of room temperature 100% methanol and incubate for exactly 5 minutes at room temperature (do not exceed 5 minutes)

• Wash cells 5 times with PBS (2 mL/wash, 2 minutes each)

• Add 2 mL of blocking solution (PBS containing 10% FBS) and incubate for 20 minutes at room temperature

• Remove blocking solution and add 1 mL of PBS/10% FBS containing FITC-conjugated antibody at 1:500 dilution

• Incubate for 1 hour at room temperature in the dark

• Wash cells 2 times with PBS (5 minutes each)

• Observe cells using a fluorescence microscope equipped with an appropriate FITC filter

This protocol may need optimization depending on cell type, target protein abundance, and specific experimental requirements.

Can USP17L6P Antibody, FITC conjugated be used for Western blot analysis?

Recommended approach:

• Perform standard protein separation by SDS-PAGE and transfer to a membrane

• Block the membrane using standard blocking buffer

• Incubate with the FITC-conjugated USP17L6P Antibody (typically at 1:1000 to 1:5000 dilution)

• Wash according to standard Western blot protocols

• Use a HRP- or AP-conjugated secondary antibody that recognizes rabbit IgG

• Develop using appropriate chemiluminescent or colorimetric detection reagents

Alternatively, for direct fluorescence detection:

• After incubation with the FITC-conjugated primary antibody and washing

• Visualize using a fluorescence imaging system compatible with FITC detection (488 nm excitation)

• Note that this approach may have lower sensitivity than chemiluminescent detection

What are common issues encountered with FITC-conjugated antibodies and their solutions?

The following table outlines common problems encountered with FITC-conjugated antibodies in immunofluorescence experiments and their potential solutions:

When troubleshooting, always include appropriate positive and negative controls to validate your experimental system and antibody performance .

How can I distinguish between specific and non-specific binding when using USP17L6P Antibody, FITC conjugated?

To distinguish between specific and non-specific binding:

• Include essential controls:

  • Negative control: Samples without primary antibody treatment but with all other steps

  • Isotype control: Use a FITC-conjugated rabbit IgG polyclonal antibody with irrelevant specificity

  • Blocking peptide control: Pre-incubate the antibody with excess immunizing peptide before staining (if available)

• Optimize antibody concentration:

  • Titrate the antibody concentration to determine the optimal signal-to-noise ratio

  • Use the highest dilution that still gives detectable specific signal

• Improve blocking conditions:

  • Extend blocking time beyond the standard 20 minutes if necessary

  • Try alternative blocking agents (5% BSA, normal serum from the same species as the secondary antibody)

  • Add 0.1-0.3% Triton X-100 to permeabilize cells if the target is intracellular

• Pattern analysis:

  • Evaluate if the observed staining pattern is consistent with the known subcellular localization of USP17L6P

  • Non-specific binding often appears as diffuse background rather than distinct localization patterns

• Validation using knockdown or knockout systems:

  • For definitive confirmation, compare staining in cells with normal versus reduced expression of USP17L6P (using siRNA or CRISPR methods)

How does USP17L6P function in the ubiquitin pathway and what are key research applications?

USP17L6P (Ubiquitin carboxyl-terminal hydrolase 17-like protein 6) functions as a deubiquitinating enzyme (DUB) that specifically cleaves ubiquitin from proteins that have been modified with ubiquitin. This enzyme belongs to the USP (Ubiquitin-Specific Protease) family, which plays crucial roles in protein degradation, cell cycle regulation, and various signaling pathways.

Key research applications include:

• Protein degradation studies: Investigating how USP17L6P regulates protein turnover by deubiquitinating specific substrates

• Cell cycle research: Examining its role in cell cycle progression and mitosis

• Cancer research: Exploring potential associations between USP17L6P expression/activity and tumor development or progression

• Drug discovery: Screening for inhibitors of USP17L6P activity as potential therapeutic agents

• Protein-protein interaction studies: Identifying binding partners and substrates of USP17L6P

The FITC-conjugated antibody enables direct visualization of the enzyme in cellular contexts, facilitating the study of its subcellular localization and potential relocalization under various physiological or pathological conditions .

What experimental considerations are important when combining USP17L6P detection with other fluorescent markers?

When designing multiplex immunofluorescence experiments combining FITC-conjugated USP17L6P antibody with other fluorescent markers:

• Spectral considerations:

  • FITC has excitation/emission maxima at approximately 495/519 nm

  • Select complementary fluorophores with minimal spectral overlap (e.g., Texas Red, Cy5, or Alexa Fluor 647)

  • If using confocal microscopy, ensure sequential scanning to minimize bleed-through

• Antibody compatibility:

  • When using multiple primary antibodies, they should be from different host species (e.g., rabbit anti-USP17L6P with mouse anti-other target)

  • If multiple antibodies from the same species are necessary, use specialized sequential staining protocols

• Sample preparation:

  • Ensure fixation method is compatible with all target epitopes

  • Optimize permeabilization conditions for access to all subcellular compartments of interest

• Controls for multiplexing:

  • Single-color controls to establish appropriate acquisition settings

  • Fluorescence minus one (FMO) controls to determine thresholds for positive staining

  • Crossreactivity controls to ensure secondary antibodies don't cross-react

• Quantitative analysis:

  • For co-localization studies, use appropriate statistical measures (Pearson's correlation, Manders' overlap coefficient)

  • Consider 3D acquisition for improved spatial resolution of interaction sites

These considerations are essential for generating reliable data in complex immunofluorescence experiments involving USP17L6P detection alongside other cellular markers .

How can researchers optimize fixation methods for USP17L6P detection in different cellular compartments?

Optimizing fixation methods is critical for preserving both antigen accessibility and cellular architecture when detecting USP17L6P in different cellular compartments:

• Fixative selection based on subcellular localization:

  • Methanol fixation: Provides good nuclear protein detection, but may extract some cytoplasmic proteins and disrupt membrane structures. Use 100% methanol at -20°C for 5-10 minutes.

  • Paraformaldehyde (PFA) fixation: Better preserves cellular morphology and membrane proteins. Use 4% PFA for 10-15 minutes at room temperature, followed by permeabilization with 0.1-0.5% Triton X-100 for cytoplasmic and nuclear proteins.

  • Combination fixation: For comprehensive detection, consider 2% PFA for 10 minutes followed by methanol (-20°C) for 5 minutes.

• Epitope retrieval considerations:

  • If initial detection is suboptimal, consider mild antigen retrieval methods:

    • Heat-mediated retrieval in citrate buffer (pH 6.0)

    • Enzymatic retrieval using proteases at very low concentrations

    • Note: Always validate that retrieval methods do not adversely affect tissue morphology

• Permeabilization optimization:

  • For nuclear proteins: 0.5% Triton X-100 for 10 minutes

  • For cytoplasmic proteins: 0.1-0.2% Triton X-100 for 5 minutes

  • For membrane-associated proteins: 0.1% saponin (maintains membrane integrity better than Triton)

• Buffer system considerations:

  • PBS-based systems (pH 7.4) are standard but may not be optimal for all applications

  • Consider HEPES-based buffers (pH 7.2-7.4) for better preservation of certain cellular structures

• Validation approach:

  • Test multiple fixation protocols in parallel

  • Compare staining patterns with published data on USP17L6P localization

  • Confirm specificity using appropriate controls (described in FAQ 3.2)

  • Document optimal conditions for reproducibility

What are appropriate quantification methods for immunofluorescence data with FITC-conjugated antibodies?

Quantification of immunofluorescence data using FITC-conjugated antibodies requires appropriate methods to ensure reliability and reproducibility:

• Intensity-based measurements:

  • Mean fluorescence intensity (MFI) within regions of interest (ROIs)

  • Integrated density (product of area and mean gray value)

  • Corrected total cell fluorescence (CTCF) = Integrated density - (Area of selected cell × Mean fluorescence of background)

• Confocal microscopy considerations:

  • Z-stack acquisition for 3D intensity measurements

  • Maximum intensity projections for comprehensive visualization

  • Optical section thickness optimization for target size

• High-content imaging approaches:

  • Automated multi-parameter analysis (intensity, area, shape, texture)

  • Machine learning algorithms for pattern recognition

  • Cell-by-cell analysis for population heterogeneity assessment

• Statistical analysis recommendations:

  • Normalize data to account for experiment-to-experiment variations

  • Use appropriate statistical tests based on data distribution

  • Report both biological and technical replicates

  • Include sample size and power calculations

• Standardization practices:

  • Use fluorescent calibration beads for instrument calibration

  • Include exposure time and gain settings in methods reporting

  • Maintain consistent acquisition parameters across comparative samples

  • Apply flat-field correction for uniform illumination

• Software options for quantification:

  • ImageJ/FIJI with appropriate plugins for immunofluorescence analysis

  • CellProfiler for automated pipeline development

  • Commercial software packages compatible with specific microscopy platforms

Each of these approaches has specific strengths, and the choice should be guided by the research question and experimental design .

How should researchers report USP17L6P localization patterns in scientific publications?

When reporting USP17L6P localization patterns in scientific publications, researchers should adhere to the following best practices:

• Comprehensive methodology documentation:

  • Detailed antibody information: Catalog number (CSB-PA757690LC01HU), lot number, supplier

  • Complete fixation and permeabilization protocols with exact concentrations and timings

  • Antibody dilution, incubation time, and temperature

  • Blocking reagents and conditions

  • Image acquisition parameters (microscope type, objective, filter sets, exposure settings)

• Image presentation standards:

  • Include scale bars on all images

  • Present representative images alongside quantification from multiple experiments

  • Show entire cells/fields alongside magnified regions of interest

  • Use consistent brightness/contrast settings for comparative images

  • Include positive and negative controls in figure panels

• Co-localization documentation:

  • When claiming co-localization with other markers, provide:

    • Individual channel images

    • Merged images

    • Quantitative co-localization metrics

    • Line scan analyses across regions of interest

• Subcellular localization characterization:

  • Specify observed patterns (e.g., nuclear, cytoplasmic, membrane-associated, vesicular)

  • Compare with established subcellular markers

  • Document any treatment-induced changes in localization patterns

  • Consider super-resolution techniques for detailed localization claims

• Quantification reporting:

  • Present data from multiple biological replicates (minimum n=3)

  • Show individual data points alongside means/medians

  • Include appropriate statistical analyses

  • Provide both raw and normalized data where relevant

• Replication and validation:

  • Confirm key findings with alternative detection methods

  • Address potential fixation artifacts by comparing multiple fixation methods

  • Consider orthogonal approaches (biochemical fractionation, proximity ligation)

These reporting standards ensure transparency, reproducibility, and scientific rigor in USP17L6P localization studies .