STYX Antibody, FITC conjugated

What is STYX and why is a FITC-conjugated antibody useful for its detection?

STYX is a catalytically inactive phosphatase that functions as a nuclear anchor for MAPK1/MAPK3 (ERK1/ERK2) and modulates cell-fate decisions and cell migration through spatiotemporal regulation of these kinases . By binding to the F-box of FBXW7, STYX prevents the assembly of FBXW7 into the SCF E3 ubiquitin-protein ligase complex, thereby inhibiting degradation of its substrates .

FITC-conjugated antibodies against STYX allow direct visualization of this protein in various experimental settings without requiring secondary antibody incubation steps. FITC has high fluorescence with excitation and emission peak wavelengths at approximately 495nm and 525nm, providing a bright yellow-green signal ideal for fluorescence microscopy and flow cytometry applications . This direct conjugation enables more streamlined experimental protocols, particularly in multicolor detection systems.

What applications are suitable for STYX Antibody, FITC conjugated?

Based on available product information and related FITC-conjugated antibodies, STYX Antibody, FITC conjugated can be utilized in multiple research applications:

• Immunofluorescence (IF/ICC): For cellular localization studies, particularly useful for examining STYX's role as a nuclear anchor for MAPK proteins

• Flow Cytometry: For quantitative analysis of STYX expression in cell populations

• Immunohistochemistry (IHC): For tissue-level expression analysis

• FACS (Fluorescence-Activated Cell Sorting): For isolation of cells expressing STYX

For example, in flow cytometry applications, researchers can follow protocols similar to those described for other FITC-conjugated antibodies, where cells are fixed, permeabilized (as STYX is intracellular), incubated with the FITC-conjugated STYX antibody, and then analyzed using standard flow cytometry equipment with appropriate filters for FITC detection .

How should STYX Antibody, FITC conjugated be stored and handled to maintain optimal performance?

Proper storage and handling of FITC-conjugated antibodies is critical to maintain their performance. For STYX Antibody, FITC conjugated, follow these guidelines:

• Storage temperature: Store at 2-8°C (refrigerated) and do not freeze, as noted for other FITC-conjugated antibodies

• Light sensitivity: Protect from continuous light exposure, which can cause gradual loss of fluorescence

• Long-term storage: For extended periods, store in aliquots at -20°C to -70°C under sterile conditions after reconstitution

• Freeze-thaw cycles: Avoid repeated freeze-thaw cycles which can degrade antibody performance

• Buffer conditions: Typically stored in PBS pH 7.4 with stabilizers such as BSA (1-20 mg/ml), sodium azide (0.02-0.03%) and often glycerol (20-50%)

When working with the antibody, minimize exposure to light during all experimental steps and handle at room temperature for the shortest time possible to preserve fluorescence intensity.

What controls should be included when using STYX Antibody, FITC conjugated?

Including appropriate controls is essential for valid and interpretable results when using FITC-conjugated STYX antibodies:

• Negative controls:

  • Isotype control: Use a FITC-conjugated antibody of the same isotype (e.g., rabbit IgG-FITC if using rabbit anti-STYX) to assess non-specific binding

  • Unstained control: Cells processed identically but without any antibody to establish autofluorescence baseline

  • Secondary-only control (for indirect methods): If using an indirect detection method alongside the direct FITC conjugate

• Positive controls:

  • Cells/tissues known to express STYX (based on literature)

  • Recombinant STYX protein for antibody validation

• Specificity controls:

  • STYX knockdown/knockout samples to confirm signal specificity

  • Blocking peptide competition to verify target-specific binding

For flow cytometry experiments specifically, as demonstrated with other FITC-conjugated antibodies, researchers should obtain and analyze representative data from at least 10,000 cells per sample to ensure statistical significance .

How does FITC conjugation affect antibody performance compared to unconjugated versions?

FITC conjugation can influence antibody characteristics in several important ways:

• Binding affinity: FITC labeling is negatively correlated with binding affinity for target antigens . Higher FITC-labeling indices typically result in reduced binding affinity to the target antigen.

• Sensitivity vs. specificity trade-off: While antibodies with higher FITC-labeling indices tend to show increased sensitivity, they are also more likely to produce non-specific staining .

• Biological activity: FITC conjugation to proteins is relatively simple and usually does not significantly alter the biological activity of the labeled protein .

• Application differences: FITC-conjugated antibodies eliminate the need for secondary antibodies in fluorescence-based applications, reducing experimental time and potential cross-reactivity, but may have reduced signal compared to indirect detection methods using unconjugated primary antibodies with fluorescently-labeled secondary antibodies.

Selection of FITC-labeled antibodies should therefore involve careful consideration of labeling index to minimize decreases in binding affinity while achieving appropriate sensitivity and specificity for the intended application .

How can STYX Antibody, FITC conjugated be optimized for studying stress granule formation?

STYX plays a significant role in stress granule assembly, making FITC-conjugated STYX antibodies valuable tools for studying this process. Based on research findings:

STYX inhibits stress granule assembly by regulating G3BP-1, a key stress granule component . When designing experiments to study this phenomenon:

• Co-localization studies:

  • Use FITC-conjugated STYX antibody in combination with differently labeled antibodies against stress granule markers (e.g., G3BP-1)

  • Implement double-staining protocols using anti-G3BP-1 and anti-FLAG sera as demonstrated in previous STYX research

  • Quantify co-localization using appropriate imaging software

• Experimental conditions:

  • Compare stress granule formation between cells expressing wild-type STYX and active mutant STYX

  • Include positive controls by inducing stress granules with arsenite or other stressors

  • Evaluate how STYX-FITC localization changes during stress response

• Quantification approach:

  • Score cells for stress granule formation (percentage of cells with stress granules)

  • Measure stress granule size and number per cell

  • Compare wild-type versus mutants, as research has shown "~23% of cells expressing the active mutant assembled stress granules, whereas MK-STYX was comparable to the control"

• Protocol refinement:

  • Optimize fixation and permeabilization conditions to preserve stress granule integrity

  • Implement appropriate blocking to minimize background fluorescence

  • Use low antibody concentrations (1:50-1:200 dilution range) for optimal signal-to-noise ratio

These approaches allow researchers to investigate how STYX regulates stress granule dynamics, particularly through its interaction with G3BP-1 and influence on cellular stress responses.

What are best practices for using STYX Antibody, FITC conjugated in multiplexed flow cytometry?

When incorporating FITC-conjugated STYX antibody into multiplexed flow cytometry panels, consider these critical factors:

• Spectral compatibility:

  • FITC emits at ~525nm (green), so select other fluorophores with minimal spectral overlap

  • Compatible fluorophores commonly used alongside FITC include TRITC, Cyanine 3, Texas Red, and Cyanine 5

  • Implement proper compensation controls for each fluorophore in the panel

• Panel design:

  • Assign FITC to targets with intermediate-to-high expression levels since FITC has moderate brightness

  • Reserve brighter fluorophores (PE, APC) for low-abundance targets

  • Include FMO (Fluorescence Minus One) controls for accurate gating

• Staining protocol optimization:

  • For intracellular STYX detection, use appropriate fixation and permeabilization reagents

• Data analysis considerations:

  • Acquire at least 10,000 cells per sample for statistical significance

  • Use appropriate gating strategies to isolate populations of interest

  • Apply consistent analysis parameters across experimental and control samples

• Quality control:

  • Include unstained, single-stained, and isotype controls

  • Monitor fluorescence stability throughout data acquisition

  • Consider viability dyes compatible with fixed cells if needed

These guidelines help ensure reliable and interpretable results when using FITC-conjugated STYX antibody in complex flow cytometry panels.

How does FITC labeling index affect STYX antibody binding affinity and specificity?

The FITC labeling index (number of FITC molecules per antibody) significantly impacts antibody performance. Based on research findings:

There is an inverse relationship between FITC-labeling index and binding affinity for target antigens . For STYX antibody research, this relationship has important implications:

• Binding affinity considerations:

  • Higher FITC-labeling indices reduce binding affinity to STYX protein

  • Researchers should select antibodies with optimal FITC:antibody ratios for their specific application

  • When quantitative analyses are required, lower labeling indices may be preferable despite reduced brightness

• Sensitivity versus specificity trade-offs:

  • Antibodies with higher FITC-labeling indices tend to be more sensitive but more likely to produce non-specific staining

  • This creates an important balance to consider when selecting reagents for different applications

  • For co-localization studies or tissues with high background, lower labeling indices may be preferable

• Experimental validation approaches:

  • Compare multiple antibody lots with different labeling indices

  • Perform titration experiments to determine optimal concentration for each application

  • Include appropriate negative controls (isotype-matched FITC-conjugated antibodies) to assess background

• Application-specific considerations:

  • For flow cytometry: Higher labeling indices may be acceptable due to compensation capabilities

  • For high-resolution microscopy: Lower labeling indices often provide better signal-to-noise ratios

  • For quantitative studies: Consistent labeling indices between experiments is critical

Based on these findings, researchers should "carefully select from several differently labeled antibodies to minimize the decrease in the binding affinity and achieve the appropriate sensitivity and interpretation" when working with FITC-conjugated STYX antibodies.

What methodological approaches can enhance signal-to-noise ratio when using FITC-conjugated STYX antibodies in challenging samples?

Achieving optimal signal-to-noise ratios with FITC-conjugated STYX antibodies in technically challenging samples requires specialized approaches:

• Sample preparation optimization:

  • Fixation method selection: For STYX detection, 4% paraformaldehyde fixation for 15-20 minutes preserves morphology while maintaining epitope accessibility

  • Antigen retrieval: For FFPE tissues, citrate buffer (pH 6.0) heat-mediated retrieval may enhance STYX epitope exposure

  • Permeabilization: Optimize detergent concentration (0.1-0.3% Triton X-100) and duration (5-15 minutes) based on cell/tissue type

• Blocking strategies:

  • Extend blocking time to 1-2 hours at room temperature for high-background samples

• Signal enhancement techniques:

  • Anti-FITC antibody amplification: Using anti-FITC antibodies conjugated to brighter fluorophores can enhance signal

  • Sequential antibody layering: Apply unconjugated STYX antibody followed by FITC-conjugated secondary, then anti-FITC tertiary antibody

  • Tyramide signal amplification: Compatible with FITC detection systems for significant signal enhancement

• Background reduction methods:

  • Autofluorescence quenching: Pretreat samples with 0.1-1% sodium borohydride or commercial reagents

  • Sudan Black B (0.1-0.3% in 70% ethanol): Reduces lipofuscin-based background

  • Additional washing steps: Implement extended wash protocols with 0.05-0.1% Tween-20 in PBS

• Imaging considerations:

  • Confocal microscopy: Reduces out-of-focus fluorescence

  • Spectral unmixing: Separates FITC signal from autofluorescence

  • Deconvolution algorithms: Enhance signal clarity post-acquisition

These methodological refinements significantly improve detection of STYX using FITC-conjugated antibodies in samples that present technical challenges such as high autofluorescence or limited target abundance.

How can STYX Antibody, FITC conjugated be effectively used to investigate MAPK signaling pathway dynamics?

STYX functions as a nuclear anchor for MAPK1/MAPK3 (ERK1/ERK2) and modulates cell-fate decisions through spatiotemporal regulation of these kinases . FITC-conjugated STYX antibodies provide valuable tools for investigating these signaling dynamics:

• Co-localization studies with MAPK pathway components:

  • Design dual-labeling experiments using FITC-conjugated STYX antibody and differently labeled antibodies against MAPK pathway components

  • Implement quantitative co-localization analysis to measure association between STYX and MAPK proteins

  • Track changes in localization patterns following pathway stimulation or inhibition

• Live-cell imaging approaches:

• Quantitative analysis of STYX-MAPK interactions:

  • Flow cytometry can quantify STYX expression levels in different cellular compartments

  • Implement appropriate gating strategies to isolate populations of interest and analyze at least 10,000 cells per sample

• Functional studies:

  • Use FITC-conjugated STYX antibody to correlate STYX localization with MAPK activity states

  • Combine with phospho-specific antibodies against activated MAPK proteins

  • Track changes following cell stimulation, stress response, or drug treatments

• Experimental considerations:

  • Appropriate controls: Include phosphatase inhibitors during sample preparation

  • Signal validation: Confirm specificity using STYX knockout/knockdown samples

  • Quantification: Employ digital image analysis to measure nuclear/cytoplasmic ratios of STYX and MAPK

This methodological framework enables researchers to investigate how STYX influences MAPK signaling dynamics through its role as a pseudophosphatase and scaffold protein.