STYX Antibody

What is STYX and why are antibodies against it important in research?

STYX (serine/threonine/tyrosine-interacting protein) is a pseudophosphatase that possesses the molecular determinants for binding phosphorylated substrates but lacks catalytic activity due to substitutions in the active site . Research has demonstrated that STYX plays oncogenic roles in several cancers, including gastric cancer, colorectal cancer, breast cancer, and endometrial cancer .

Antibodies against STYX are critical research tools for several reasons:

• Detection and quantification: They enable researchers to identify and measure STYX protein expression in different tissues and cell types, providing insights into its normal distribution and potential dysregulation in disease states.

• Functional studies: STYX antibodies facilitate investigations into protein-protein interactions, such as STYX binding to FBXO31 in gastric cancer, which inhibits the degradation of target proteins CyclinD1 and Snail1 .

• Diagnostic and prognostic research: As STYX has been identified as a potential diagnostic and prognostic marker for gastric cancer patients, antibodies are essential for developing and validating clinical applications .

• Mechanistic investigations: These antibodies allow researchers to explore the mechanisms through which STYX contributes to cellular processes like proliferation and migration in both normal physiology and pathological conditions.

In experimental settings, researchers typically use affinity-purified polyclonal antisera or monoclonal antibodies raised against full-length recombinant STYX protein for these applications . The specificity of these antibodies is crucial for generating reliable and reproducible results in STYX-related research.

What are the common applications of STYX antibodies in cancer research?

STYX antibodies serve multiple critical applications in cancer research, particularly in investigating its role as an oncogenic factor:

For Western blotting applications, protocols typically include blocking membranes with 5% nonfat dry milk before probing with STYX antibody, followed by detection with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence . Optimization of antibody dilution, incubation conditions, and detection systems is essential for each specific application.

How do researchers validate the specificity of STYX antibodies?

Validating the specificity of STYX antibodies is crucial for ensuring reliable research results, particularly given the existence of STYX pseudogenes. Researchers employ several complementary approaches:

• Positive and negative controls:

  • Positive controls include samples known to express STYX (e.g., testis tissue in mice)

  • Negative controls include tissues from STYX knockout models

  • Comparing antibody reactivity between these controls helps confirm specificity

• Knockdown/overexpression validation:

  • Analyzing samples with experimentally manipulated STYX expression levels

  • Comparing Western blot signals between wild-type cells and those transfected with STYX siRNAs or overexpression vectors

  • A specific antibody will show decreased signal in knockdown samples and increased signal in overexpression samples

• Cross-technique verification:

  • Confirming that protein detection (Western blot) correlates with mRNA expression (qRT-PCR)

  • For example, if STYX protein levels detected by the antibody decrease after siRNA treatment, STYX mRNA levels should show a corresponding decrease

  • This multi-method approach increases confidence in antibody specificity

• Multiple antibodies targeting different epitopes:

  • Using multiple antibodies that recognize different regions of the STYX protein

  • Concordant results with different antibodies increase confidence in specificity

• Peptide competition assays:

  • Pre-incubating the antibody with purified STYX protein or peptide

  • This should block specific binding sites on the antibody

  • Subsequent loss of signal in immunoassays confirms specificity

In studies of STYX in gastric cancer, researchers validated antibody specificity by showing consistent results between protein detection (Western blot) and mRNA expression (qRT-PCR) following STYX knockdown or overexpression . This multipronged approach to validation ensures that observed effects are truly attributable to STYX rather than to cross-reactivity with related proteins.

What types of STYX antibodies are available for research purposes?

Several types of STYX antibodies are available for research purposes, each with distinct characteristics suitable for different experimental applications:

• Polyclonal antibodies:

  • Produced by immunizing animals (typically rabbits, goats, or sheep) with purified STYX protein or peptides

  • Recognize multiple epitopes on the STYX protein

  • Advantages: High sensitivity, robust for various applications

  • Example: Affinity-purified polyclonal antisera raised against full-length recombinant mouse STYX

  • Methodological note: These are typically used at dilutions of 1:500 to 1:2000 for Western blotting

• Monoclonal antibodies:

  • Produced from a single B-cell clone, recognizing a single epitope

  • Advantages: High specificity, consistent lot-to-lot performance

  • Particularly valuable for distinguishing between STYX and its pseudogenes (STYX-rs1 and STYX-ps1)

  • Methodological note: Often used at 1:1000 to 1:5000 dilutions for Western blotting

• Application-specific antibodies:

  • Antibodies validated for specific techniques such as:

    • Western blot-specific antibodies

    • Immunohistochemistry/Immunofluorescence-optimized antibodies

    • Immunoprecipitation-validated antibodies

    • Flow cytometry-compatible antibodies

  • Using application-validated antibodies significantly improves experimental success rates

• Species-specific antibodies:

  • Antibodies targeting human STYX, mouse STYX, or other species

  • Important consideration given the species-specific expression patterns of STYX (e.g., primarily in testes in mice)

  • Critical for cross-species studies or when working with different model organisms

• Domain-specific antibodies:

  • Antibodies targeting specific functional domains of STYX

  • Useful for investigating structure-function relationships

  • May be used to block specific interactions in functional studies

When selecting a STYX antibody, researchers should consider the specific application, whether epitope accessibility might be affected by experimental conditions, and the validation data available for the antibody. For critical experiments, testing multiple antibodies or validating a new antibody against previously characterized ones is recommended.

What are the optimal conditions for using STYX antibodies in Western blot applications?

Achieving optimal results with STYX antibodies in Western blot applications requires careful attention to several methodological parameters:

• Sample preparation:

  • Effective protein extraction: Total protein can be recovered from tissues using reagents like TRIzol, following manufacturer's protocols

  • Appropriate lysis buffers: For STYX, options include Nonidet P-40 buffer (150 mM NaCl/1% Nonidet P-40/50 mM Tris, pH 8.0), high salt buffer (500 mM NaCl), or RIPA buffer (additional 0.5% deoxycholate and 0.1% SDS)

  • Protease inhibitors: Include Complete protease inhibitors to prevent protein degradation during extraction

  • Sample standardization: Equal protein loading (typically 20-50 μg per lane) confirmed by BCA or Bradford assay

• Gel electrophoresis and transfer:

  • SDS-PAGE conditions: 10-12% polyacrylamide gels are typically suitable for resolving STYX (molecular weight ~25-30 kDa)

  • Transfer parameters: 100V for 1-2 hours or 30V overnight in Tris-glycine buffer with 20% methanol

  • Transfer verification: Use Ponceau S staining to confirm successful protein transfer

• Blocking conditions:

  • Optimal blocking: 5% nonfat dry milk in TBS or PBS with 0.1% Tween-20 (TBST/PBST) for 1 hour at room temperature

  • Alternative blocking: 3-5% BSA in TBST may be used for phospho-specific applications

• Primary antibody incubation:

  • Dilution: Typically 1:500 to 1:2000 for polyclonal anti-STYX antibodies in blocking buffer

  • Incubation: Overnight at 4°C with gentle agitation provides optimal sensitivity and specificity

  • Washing: 3-5 washes with TBST/PBST for 5-10 minutes each after primary antibody incubation

• Secondary antibody and detection:

  • Secondary antibody: Horseradish peroxidase-conjugated anti-IgG antibodies at 1:5000-1:10000 dilution

  • Incubation: 1 hour at room temperature

  • Washing: 3-5 thorough washes with TBST/PBST

  • Detection: Enhanced chemiluminescence (ECL) systems are effective for detecting STYX

  • Exposure: Start with short exposures (30 seconds) and increase as needed

• Controls and troubleshooting:

  • Positive control: Include samples known to express STYX (e.g., gastric cancer cell lines for human STYX or mouse testis for mouse STYX)

  • Loading control: Probe for housekeeping proteins like GAPDH, β-actin, or α-tubulin

  • Stripping and reprobing: If needed, gentle stripping buffers can be used to reprobe membranes

These optimized conditions have been successfully employed in studies investigating STYX expression in gastric cancer cells and mouse testis, yielding specific detection with minimal background .

How should STYX antibodies be stored and handled to maintain their efficacy?

Proper storage and handling of STYX antibodies are critical for maintaining their performance and extending their usable lifespan. Here are methodological recommendations for optimal antibody management:

• Storage temperature:

  • Long-term storage: Store antibodies at -20°C or -80°C in small aliquots to avoid repeated freeze-thaw cycles

  • Working solutions: Store at 4°C for up to one month

  • Avoid storing antibodies at room temperature for extended periods

• Aliquoting strategy:

  • Upon receipt, divide antibodies into small single-use aliquots (typically 10-20 μL)

  • Use sterile microcentrifuge tubes for aliquoting

  • Label tubes with antibody name, lot number, date, and dilution if pre-diluted

  • This practice minimizes freeze-thaw cycles, which can denature antibodies and reduce activity

• Freeze-thaw management:

  • Limit freeze-thaw cycles to a maximum of 5 times

  • Thaw antibodies slowly on ice rather than at room temperature

  • Return to storage promptly after use

  • Record the number of freeze-thaw cycles on the tube label

• Dilution and working solution preparation:

  • Dilute antibodies in freshly prepared, cold buffers

  • For STYX antibodies in Western blot applications, 5% nonfat dry milk in TBST is typically used

  • For immunoprecipitation, dilute in the appropriate lysis buffer (e.g., Nonidet P-40 buffer, high salt buffer, or RIPA buffer)

  • Consider adding sodium azide (0.02%) to working dilutions stored at 4°C to prevent microbial growth

• Contamination prevention:

  • Use clean pipette tips for each handling

  • Work in a clean environment to avoid contaminants

  • Consider adding preservatives for diluted antibodies stored for more than a week

• Stability indicators:

  • Monitor antibody performance over time using positive controls

  • Watch for decreased signal intensity or increased background

  • Increased concentration requirements may indicate deterioration

Following these methodological practices ensures consistent performance of STYX antibodies across experiments and maximizes the value of these research reagents. Proper documentation of storage conditions, freeze-thaw cycles, and performance characteristics helps maintain antibody quality over time.

How can STYX antibodies be used to investigate the interaction between STYX and FBXO31?

Investigating the interaction between STYX and FBXO31 requires sophisticated methodological approaches using STYX antibodies. Based on research showing that STYX binds to the F-box of FBXO31 and inhibits its function in targeting proteins for degradation , several techniques can be employed:

• Co-immunoprecipitation (Co-IP) protocols:

  • Standard Co-IP:

    • Lyse cells in appropriate buffer (Nonidet P-40 buffer, high salt buffer, or RIPA buffer)

    • Pre-clear lysates with protein-A agarose for 16h at 4°C to reduce non-specific binding

    • Incubate cleared lysates with anti-STYX antibodies for 1h at 4°C

    • Capture immune complexes on protein-A agarose for 1h at 4°C

    • Wash complexes 4 times with lysis buffer and elute for SDS-PAGE analysis

    • Probe Western blots with anti-FBXO31 antibodies to detect interaction

  • Reverse Co-IP:

    • Perform the same procedure using anti-FBXO31 antibodies for immunoprecipitation

    • Detect STYX in the precipitated complex using anti-STYX antibodies

    • Compare results from both directions to confirm interaction

• Domain-specific interaction mapping:

  • Generate constructs expressing FBXO31 with mutations or deletions in the F-box domain

  • Perform Co-IP with anti-STYX antibodies

  • Compare binding efficiency to wild-type FBXO31

  • This approach can map the specific regions required for interaction

• Functional impact analysis:

  • Immunoprecipitate FBXO31 from cells with and without STYX overexpression

  • Analyze the binding of FBXO31 to its targets (CyclinD1 and Snail1) using specific antibodies

  • This demonstrates how STYX affects FBXO31's ability to bind its substrates

• Proximity ligation assay (PLA):

  • Fix cells on microscope slides and permeabilize

  • Incubate with primary antibodies against both STYX and FBXO31 (from different species)

  • Add species-specific PLA probes with attached oligonucleotides

  • When proteins are in close proximity (<40 nm), oligonucleotides can interact

  • Amplify and detect signal using fluorescent probes

  • This technique visualizes protein interactions in situ with subcellular resolution