SHOC2 Antibody, FITC conjugated

What is SHOC2 and why is it an important research target?

SHOC2 functions as a core component of the SHOC2-MRAS-PP1c (SMP) holophosphatase complex that regulates activation of the MAPK pathway. As a scaffolding protein within this complex, SHOC2 facilitates the specific dephosphorylation of inhibitory phosphorylation sites on RAF kinases, including 'Ser-259' of RAF1 kinase, 'Ser-365' of BRAF kinase, and 'Ser-214' of ARAF kinase, thereby stimulating their kinase activities . The SMP complex enhances both the dephosphorylation activity and substrate specificity of PP1c, making SHOC2 a critical regulatory node in signal transduction . Mutations in SHOC2 are associated with Noonan syndrome-like with loose anagen hair (NSLAH), a condition characterized by macrocephaly, hypertelorism, palpebral ptosis, and other developmental features .

What are the key specifications to consider when selecting a FITC-conjugated SHOC2 antibody?

When selecting a FITC-conjugated SHOC2 antibody, researchers should consider:

• Immunogen region: Various antibodies target different regions of SHOC2, such as amino acids 36-120/582, 3-89, or the C-terminal domain

• Host organism: Typically rabbit for polyclonal antibodies

• Validated applications: Confirm the antibody has been tested for your specific application (WB, IF, IHC-P, IHC-F, ICC)

• Species reactivity: Verify reactivity with your experimental model (human, mouse, rat)

• Storage buffer composition: Most contain glycerol (typically 50%) with preservatives like Proclin300 or sodium azide

• Storage conditions: Typically -20°C with aliquoting recommended to avoid freeze-thaw cycles

How do FITC-conjugated SHOC2 antibodies differ from unconjugated versions in research applications?

FITC-conjugated SHOC2 antibodies offer direct fluorescence detection capability without requiring secondary antibodies, which provides several methodological advantages:

• Simplified protocols: Elimination of secondary antibody incubation steps reduces experiment time and potential background issues

• Direct visualization: Enables immediate detection in immunofluorescence applications with excitation/emission appropriate for FITC (typically 495nm/519nm)

• Multiplexing capability: Facilitates co-staining with antibodies raised in the same host species but conjugated to different fluorophores

• Application limitations: While excellent for IF/ICC applications, FITC-conjugated antibodies are not suitable for applications requiring enzymatic detection (like conventional WB or IHC-DAB)

• Photostability considerations: FITC is more susceptible to photobleaching than some alternative fluorophores, requiring appropriate controls and imaging protocols

What are the recommended protocols for using FITC-conjugated SHOC2 antibodies in immunofluorescence applications?

For optimal results in immunofluorescence applications with FITC-conjugated SHOC2 antibodies:

Cell Preparation and Fixation:

• Grow cells on glass-bottom dishes until desired confluence

• Wash cells with Ca²⁺, Mg²⁺-free phosphate buffered saline (CMF-PBS)

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 for 5-10 minutes

Staining Protocol:

• Block with 1% BSA in PBS for 30-60 minutes

• Apply FITC-conjugated SHOC2 antibody at recommended dilution (typically 1:50-1:200) in blocking buffer

• Incubate for 1-2 hours at room temperature or overnight at 4°C in a humidified chamber protected from light

• Wash 3-5 times with PBS for 5 minutes each

• Counterstain nuclei with DAPI if desired

• Mount with anti-fade mounting medium

Imaging Considerations:

• Use appropriate filter sets for FITC (excitation: ~495nm, emission: ~519nm)

• Minimize exposure time to reduce photobleaching

• Include appropriate controls (secondary-only, isotype control)

How can SHOC2 antibodies be utilized to study protein-protein interactions within the SHOC2 complex?

SHOC2 antibodies are valuable tools for investigating protein-protein interactions within the SHOC2 scaffolding complex:

Co-Immunoprecipitation Approach:

• Lyse cells in appropriate buffer (containing protease and phosphatase inhibitors)

• Pre-clear lysate with protein A/G beads

• Incubate lysate with SHOC2 antibody (non-FITC versions are preferred for IP applications)

• Capture antibody-protein complexes with protein A/G beads

• Wash extensively to remove non-specific binding

• Elute and analyze by Western blot for interacting partners

Research Applications:

• High-affinity SHOC2 antibodies have been shown to efficiently immunoprecipitate known SHOC2 interacting partners, making them powerful tools for studying the SHOC2 scaffolding complex

• Single-domain antibodies with nanomolar binding affinities (Kd values of 14.4 nM ± 0.4 and 516 nM ± 12) have proven particularly effective for analyzing complex assembly

• Different antibodies targeting specific epitopes can help map binding regions and functional domains within SHOC2

What dilutions and controls are recommended for FITC-conjugated SHOC2 antibody applications?

Recommended Dilutions by Application:

Essential Controls:

• Negative controls:

  • Secondary antibody-only (for unconjugated antibodies)

  • Isotype control antibody at same concentration

  • SHOC2 knockout or knockdown samples

• Positive controls:

  • Cell lines with verified SHOC2 expression (MCF-7, Jurkat, U-251)

  • Tissues with known SHOC2 expression (brain, kidney, testis)

• Validation approaches:

  • Correlation with unconjugated antibody results

  • Cross-verification with antibodies targeting different SHOC2 epitopes

  • Western blot validation when possible

What are common issues with FITC-conjugated SHOC2 antibody experiments and how can they be resolved?

Problem: High Background Signal

• Causes: Insufficient blocking, excessive antibody concentration, non-specific binding

• Solutions:

  • Increase blocking time/concentration (try 5% BSA or normal serum)

  • Dilute primary antibody further (1:200-1:500 range)

  • Include 0.1% Tween-20 in wash buffers

  • Perform additional wash steps

Problem: Weak or No Signal

• Causes: Insufficient antigen exposure, antibody dilution too high, epitope masking, photobleaching

• Solutions:

  • Optimize antigen retrieval (try TE buffer pH 9.0 or citrate buffer pH 6.0 for tissue sections)

  • Increase antibody concentration

  • Extend incubation time or incubate at 4°C overnight

  • Use fresh antibody aliquot to avoid freeze-thaw damage

  • Add anti-fade reagent to mounting medium

Problem: Punctate or Uneven Staining

• Causes: Incomplete permeabilization, antibody aggregation, uneven fixation

• Solutions:

  • Optimize permeabilization (increase time or detergent concentration)

  • Centrifuge antibody before use to remove aggregates

  • Ensure consistent fixation across sample

How does phosphorylation status affect SHOC2 antibody recognition and experimental outcomes?

SHOC2 phosphorylation can significantly impact antibody recognition and experimental interpretation:

• Phosphorylation-specific detection:

  • Phospho-specific antibodies (like those recognizing phospho-Thr 507) can be used to monitor activation status of SHOC2

  • Phosphorylation at Thr 507 increases within 5 minutes of EGF stimulation and remains elevated for at least 10 minutes

  • Serum addition increases phospho-Thr 507 levels in a time-dependent manner within 2-4 hours

• Epitope masking considerations:

  • Phosphorylation near antibody binding sites can mask epitopes and reduce antibody affinity

  • Pre-treatment with phosphatase inhibitors may be necessary when studying phosphorylated SHOC2

  • Different fixation methods may preserve phospho-epitopes to varying degrees

• Functional implications:

  • Phosphorylation status affects SHOC2 protein-protein interactions and stability

  • FBXW7-mediated degradation of SHOC2 is dependent on phosphorylation at specific sites

  • Consider using phosphatase inhibitors in lysis buffers when studying SHOC2 complexes

How should FITC-conjugated SHOC2 antibodies be stored and handled to maintain optimal performance?

Storage Recommendations:

• Store at -20°C in the dark to preserve both antibody activity and fluorophore integrity

• Aliquot into small volumes upon receipt to avoid repeated freeze-thaw cycles

• For working solutions, store at 4°C protected from light for up to 2 weeks

Handling Best Practices:

• Avoid exposure to light during all handling steps to prevent photobleaching of the FITC fluorophore

• Allow the antibody to equilibrate to room temperature before opening to prevent condensation

• Centrifuge briefly before opening to collect solution at the bottom of the tube

• Use non-metallic spatulas or pipette tips when handling to prevent potential fluorophore quenching

• Return to storage promptly after use

Stability Considerations:

• Most preparations contain stabilizers and preservatives (0.01M TBS pH 7.4 with 1% BSA, 0.03% Proclin300, and 50% glycerol or similar)

• Monitor for signs of deterioration such as precipitation or significant loss of fluorescence intensity

• Validate activity periodically with positive controls if storing for extended periods

How can SHOC2 antibodies be utilized to investigate cross-talk between the RAS-ERK and mTORC1 signaling pathways?

SHOC2 antibodies are valuable tools for exploring the intricate cross-talk between major signaling pathways:

Experimental Approaches:

• Co-immunoprecipitation studies:

  • SHOC2 antibodies can pull down interaction partners like Raptor (mTORC1 component)

  • Analyze how pathway stimulation affects these interactions under various conditions

• Phosphorylation dynamics analysis:

  • Use phospho-specific antibodies alongside FITC-conjugated SHOC2 antibodies to track activation status

  • Investigate how inhibitors of either pathway affect SHOC2 phosphorylation and localization

• Degradation and stability studies:

  • The FBXW7-SHOC2-Raptor axis controls cross-talk between RAS-ERK and mTORC1 pathways

  • FBXW7 regulates both pathways by targeting SHOC2 for degradation

  • Use SHOC2 antibodies to monitor protein levels after treatment with pathway modulators

Research Findings:

• FBXW7, when ectopically expressed, can pull down endogenous SHOC2 and promote its degradation

• This degradation is proteasome-dependent and can be rescued by MG132 treatment

• Phosphorylation at Thr 507 is triggered by EGF stimulation and appears to be a prerequisite for FBXW7 binding

What role do single-domain antibodies against SHOC2 play in understanding functional mechanisms of multiprotein signaling complexes?

Single-domain antibodies (nanobodies) against SHOC2 provide unique advantages for studying complex signaling networks:

Technological Advantages:

• High binding specificity and affinity:

  • Nanomolar binding affinities (Kd values of 14.4 nM ± 0.4 and 516 nM ± 12)

  • Recognition of conformational epitopes not detected by conventional antibodies

• Superior complex assembly analysis:

  • Ability to immunoprecipitate native multiprotein complexes without disrupting critical interactions

  • Effective for analyzing transient or dynamic protein-protein associations

• Domain-specific targeting:

  • Different nanobodies recognize distinct epitopes (e.g., the region connecting N-terminal domain with C-terminal LRR domain)

  • Enables functional analysis of specific protein domains

Research Applications:

• Eight synthetic single-domain antibodies against human SHOC2 have been generated using a universal synthetic library of humanized nanobodies

• These antibodies efficiently immunoprecipitate both ectopically expressed and endogenous SHOC2 proteins

• High-affinity nanobodies have proven effective for intracellular assays and understanding how SHOC2 guides ERK1/2 signals

How can SHOC2 antibodies be utilized in investigating antimicrobial immunity mechanisms in invertebrates?

Recent research has revealed unexpected roles for SHOC2 in invertebrate immune responses, which can be studied using specialized antibodies:

Experimental Approaches:

• SHOC2-flagellin interaction studies:

  • In shrimp (Marsupenaeus japonicus), MjShoc2 recognizes bacterial flagellin (FlaA)

  • Pull-down assays with native MjShoc2 from hemocytes lysate confirm this interaction

  • Isothermal titration calorimetry (ITC) shows Kd of approximately 10 μM

• Antibacterial pathway analysis:

  • SHOC2 regulates FlaA-induced expression of antibacterial effectors MjCtl556 and MjAlf2238

  • MjCtl556 agglutinates bacteria while MjAlf2238 exhibits direct antimicrobial activity

  • Chromatin immunoprecipitation assays can determine transcriptional regulatory mechanisms

• Knockdown studies:

  • MjShoc2 knockdown leads to decreased expression of antibacterial effectors

  • This approach reveals SHOC2's contribution to transcriptional regulation in immune responses

Research Implications:

• These findings reveal a previously unknown antibacterial strategy in invertebrates

• SHOC2 antibodies can help elucidate flagellin sensing mechanisms across species

• Comparative studies between vertebrate and invertebrate SHOC2 function may reveal evolutionary conservation of immune mechanisms

What are the methodological considerations for using biolayer interferometry to characterize SHOC2 antibody binding kinetics?

Biolayer interferometry offers powerful insights into SHOC2-antibody interactions:

Experimental Protocol:

• Biosensor preparation:

  • Hydrate Protein A Biosensors in buffer A (50 mM Tris-Cl pH 7.4, 300 mM NaCl, 10 mM MgCl₂) for 50 minutes at room temperature

• Antibody loading:

  • Load sensors with antibody by incubating for approximately 1 minute

  • Use antibody concentration of 0.01mg/mL

  • Wash in buffer A for 30 seconds to remove unbound protein

• Association kinetics measurement:

  • Incubate antibody-bound biosensor with SHOC2 protein (0.03 mg/mL) for 2 minutes

  • Take data readings at 0.2-second intervals

• Dissociation kinetics measurement:

  • Immerse sensor in buffer A for 2 minutes

  • Continue data collection at 0.2-second intervals

• Controls and data analysis:

  • Include control with SHOC2 incubated with protein-A biosensor (no antibody) to rule out non-specific interactions

  • Import data into analysis software (like GraphPad Prism)

  • Determine on and off rates plus dissociation constants using nonlinear regression

Research Applications:

• This approach has successfully characterized single-domain antibodies with nanomolar affinities for SHOC2

• The method enables precise comparison between different antibodies (e.g., hs2dAb B99 with Kd 14.4 nM vs. hs2dAb B120 with Kd 516 nM)

• Kinetic parameters provide insights into antibody suitability for specific applications

What are the current limitations in SHOC2 antibody research and potential future developments?

Current Limitations:

• Epitope coverage gaps: Many commercial SHOC2 antibodies target overlapping regions, leaving some domains understudied

• Conformational epitope detection: Most antibodies struggle to recognize native conformational states

• Cross-reactivity concerns: Verification across species remains challenging due to variable conservation

• Phosphorylation-state specificity: Limited availability of antibodies recognizing specific phosphorylated forms of SHOC2

Future Developments:

• Expanded single-domain antibody libraries: Development of nanobodies targeting diverse SHOC2 epitopes

• Multi-color fluorophore conjugates: Beyond FITC to enable multiplexed detection with enhanced photostability

• Intrabody applications: Engineering antibody fragments for live-cell imaging and functional inhibition

• Phospho-specific antibody panels: Creation of comprehensive tools to monitor SHOC2 activation states

• Cross-species validated reagents: Development of antibodies with verified reactivity across multiple model organisms

How can SHOC2 antibodies contribute to understanding disease mechanisms and potential therapeutic approaches?

SHOC2 antibodies provide valuable insights into disease mechanisms with therapeutic implications:

Disease-Related Applications:

• Noonan syndrome-like with loose anagen hair (NSLAH):

  • SHOC2 mutations cause this disorder characterized by macrocephaly, hypertelorism, and other developmental features

  • Antibodies can help characterize tissue-specific expression patterns and molecular consequences of mutations

• Cancer research:

  • SHOC2 is implicated in RAF-ERK pathway dysregulation in multiple cancers

  • Antibodies can detect altered expression, localization, or phosphorylation in tumor samples

• Immune disorders:

  • Recently discovered roles in antimicrobial responses suggest involvement in immune function

  • Potential applications in studying inflammatory signaling mechanisms

Therapeutic Development:

• Single-domain antibodies show promise for therapies requiring modulation of ERK1/2-associated diseases

• Targeting specific SHOC2 interactions or conformational states could provide therapeutic selectivity

• Understanding SHOC2-mediated cross-talk between RAS-ERK and mTORC1 pathways could inform combination therapy approaches

What methodological approaches can integrate SHOC2 antibody data with other -omics techniques for systems biology insights?

Integration of SHOC2 antibody data with other -omics approaches enables comprehensive systems biology analysis:

Integrative Methodologies:

• Proteomics integration:

  • Combine immunoprecipitation with mass spectrometry to identify novel interaction partners

  • Compare SHOC2 interactomes under different cellular conditions or treatments

  • Correlate post-translational modifications with functional outcomes

• Transcriptomics correlation:

  • Link SHOC2-mediated signaling to transcriptional outputs

  • High-throughput sequencing after SHOC2 knockdown/knockout reveals downstream effectors

  • ChIP assays using SHOC2 antibodies can identify direct transcriptional targets

• Spatial proteomics:

  • Super-resolution microscopy with FITC-conjugated SHOC2 antibodies maps subcellular localization

  • Proximity ligation assays identify context-specific protein interactions

  • Correlate spatial distribution with functional outcomes

Data Integration Frameworks:

• Pathway enrichment analysis of SHOC2-associated proteins identifies functional modules

• Network analysis reveals central nodes in SHOC2-dependent signaling networks

• Machine learning approaches can predict functional consequences of SHOC2 perturbations based on multi-omics data