SOCS2 Antibody, Biotin conjugated

What is the structural composition of SOCS2 protein and its functional domains?

SOCS2 (Suppressor of Cytokine Signaling 2) consists of three main structural components: a short 32-residue N-terminal region and two major functional domains - a central SH2 domain and a SOCS box. The SOCS2-SH2 domain functions as a substrate recognition module that specifically binds to phosphorylated tyrosine residues 487 and 595 within the Growth Hormone Receptor (GHR) cytoplasmic domain . The SOCS box provides binding sites for adaptors Elongins B and C, and the Cullin 5 scaffold, which recruit RING-box protein (Rbx)2 to form an E3 ubiquitin ligase complex .

The SOCS2-SH2 domain structure is particularly notable, consisting of three β-strands flanked by two α-helices, and an additional "SOCS-specific" α-helix termed the Extended SH2 Subdomain (ESS). This domain contains a positively charged pocket (P0) that binds phosphorylated tyrosine (pTyr) and a hydrophobic patch that accommodates the third residue (+3) distal from the pTyr .

How does SOCS2 regulate growth hormone signaling and other cytokine pathways?

SOCS2 functions as a key negative regulator of growth hormone (GH) and JAK-STAT signaling pathways. The regulatory mechanism involves:

• Recognition and binding of phosphorylated proteins via its SH2 domain

• Formation of an E3 ubiquitin ligase complex (ECS complex or CRL5 complex) that mediates ubiquitination and subsequent proteasomal degradation of target proteins

• Specific regulation of growth hormone receptor (GHR) levels by mediating ubiquitination and degradation following GHR phosphorylation by JAK2

• Catalyzing ubiquitination and degradation of JAK2-phosphorylated EPOR (erythropoietin receptor)

Research has demonstrated that mice deficient in SOCS2 grow significantly larger than normal littermates, confirming SOCS2's critical role in growth regulation . Additionally, SOCS2 contributes to various biological processes including metabolism, bone formation, neuronal development, cancer, infection and other cytokine-dependent pathways .

What are the optimal experimental conditions for using SOCS2 Antibody, Biotin conjugated in ELISA assays?

For optimal ELISA performance with SOCS2 Antibody, Biotin conjugated, researchers should implement the following protocol:

• Coating Phase:

  • Use carbonate-bicarbonate buffer (pH 9.6) for coating plates with target protein

  • Incubate plates overnight at 4°C

  • Blocking with 3% BSA in PBS for 2 hours at room temperature

• Antibody Application:

  • Recommended dilution range: 1:1000 to 1:2000 in 1% BSA/PBS-T

  • Incubation period: 2 hours at room temperature with gentle shaking

  • Washing step: 5× washing with PBS-T between each step

• Detection System:

  • Apply streptavidin-HRP at 1:5000 dilution

  • Development with TMB substrate solution

  • Read absorbance at 450nm with reference at 620nm

The biotin conjugation offers superior sensitivity compared to traditional detection methods, with detection limits typically in the pg/ml range for recombinant SOCS2 protein .

How can researchers optimize immunoprecipitation protocols when using SOCS2 antibodies?

For effective immunoprecipitation of SOCS2 and its binding partners, implement the following optimized protocol:

• Cell Lysis:

  • Lyse cells in buffer containing 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and protease/phosphatase inhibitors

  • Maintain samples at 4°C throughout the procedure

  • Clear lysates by centrifugation at 14,000×g for 15 minutes

• Antibody Binding:

  • Use SOCS2 antibody at 1:100 dilution (approximately 2-4μg per sample)

  • Pre-incubate antibody with protein A/G beads for 1 hour if not using biotinylated antibody

  • For biotinylated antibody, use streptavidin-coated beads

  • Incubate lysate with antibody-bead complex overnight at 4°C with rotation

• Washing and Elution:

  • Perform stringent washing (4-5 times) with lysis buffer containing reduced detergent

  • Elute using either low pH buffer or SDS sample buffer

  • For interaction studies, milder elution conditions may preserve protein-protein interactions

When studying SOCS2 interactions with growth hormone receptor (GHR) or JAK2, consider pre-treatment of cells with growth hormone (500 ng/ml for 15 minutes) to enhance phosphorylation and binding .

How can surface plasmon resonance (SPR) be used to measure SOCS2 binding affinity to growth hormone receptor phosphopeptides?

Surface plasmon resonance provides a powerful tool for quantitatively measuring SOCS2 binding to phosphorylated receptor peptides. Based on published methodologies, researchers should:

• Surface Preparation:

  • Immobilize biotin-GSGS-GHR pY595 peptide to a Streptavidin-coated SA chip

  • Achieve immobilization levels of approximately 200-400 response units for optimal signal-to-noise ratio

• Binding Assay:

  • Prepare SOCS2 proteins at 100 nM concentration

  • Pre-incubate with titrations of GHR pY595 competitor peptide (ranging from 0.1 μM to 10 μM)

  • Flow the mixture over the chip at 30 μL/min

  • Regenerate surface between cycles with glycine-HCl pH 2.0

• Data Analysis:

  • Calculate KD values using competitive binding models

  • Compare wild-type SOCS2 with mutants (e.g., SOCS2-R73K) to understand binding mechanisms

This approach has successfully demonstrated that the SOCS2-SH2 domain has high affinity for GHR pY595, with KD values typically in the nanomolar range .

What strategies can be employed to investigate the exosite on the SOCS2-SH2 domain that enhances phosphopeptide binding?

Recent research has identified a non-canonical exosite on the SOCS2-SH2 domain that enhances binding to phosphorylated targets . To investigate this feature:

• Phage Display Library Screening:

  • Create a fusion protein consisting of the SOCS2-SH2 domain and SOCS box fused to GST, in complex with elongins B and C (GST-SOCS2 32–198-EloB/C)

  • Prior to binding, incubate the library with BSA and GST to deplete non-specific peptides

  • Perform multiple rounds of selection (5 recommended)

  • Use phage-ELISAs to detect specific binding clones

• Mutagenesis Studies:

  • Generate point mutations in the exosite region

  • Express recombinant proteins for binding studies

  • Use the SOCS2R96C mouse model to evaluate functional consequences in vivo

• Structural Analysis:

  • Employ crystallography or NMR to resolve structural features

  • Map binding interactions between SOCS2-SH2 and peptide ligands

  • Investigate the model where SOCS2 can engage phosphopeptide conventionally while simultaneously interacting with a second GHR pY595 peptide in an anti-parallel direction

This dual-binding capability provides a potential mechanism for SOCS2 interaction with both subunits in the dimerized GHR, offering new insights into SOCS2 function and regulation .

How should researchers troubleshoot non-specific binding when using SOCS2 Antibody, Biotin conjugated in Western blotting?

Non-specific binding in Western blotting with SOCS2 Antibody, Biotin conjugated can be addressed through a systematic approach:

• Blocking Optimization:

  • Test different blocking agents (BSA vs. non-fat milk)

  • Increase blocking time to 2 hours at room temperature

  • Consider adding 0.1% Tween-20 to blocking buffer

• Antibody Dilution Optimization:

  • Test a dilution series (1:500 to 1:5000)

  • Extend primary antibody incubation to overnight at 4°C

  • Perform more stringent washing between antibody steps (5× washes with PBS-T)

• Sample Preparation Considerations:

  • Ensure complete denaturation of samples

  • Add phosphatase inhibitors to preserve phosphorylation status

  • Consider using RIPA buffer with 0.1% SDS for enhanced extraction

• Controls and Validation:

  • Run positive control (recombinant SOCS2)

  • Include SOCS2 knockdown/knockout samples as negative controls

  • Pre-absorb antibody with recombinant protein to confirm specificity

The expected molecular weight for SOCS2 is approximately 22 kDa , and proper sample preparation is critical as SOCS2 expression can be highly tissue-specific and cytokine-inducible .

How can researchers differentiate between SOCS2 and other SOCS family members in immunological assays?

Differentiating between SOCS family members requires careful experimental design:

• Antibody Selection:

  • Verify antibody epitope mapping to confirm specificity for SOCS2

  • Choose antibodies raised against non-conserved regions of SOCS2

  • Consider using multiple antibodies targeting different epitopes

• Experimental Validation:

  • Perform side-by-side testing with recombinant SOCS1, SOCS2, SOCS3, and CIS proteins

  • Include knockout/knockdown controls for each SOCS family member

  • Analyze expression pattern in tissues with known differential expression

• Mass Spectrometry Confirmation:

  • Following immunoprecipitation, perform LC-MS/MS analysis

  • Identify unique peptides specific to SOCS2

  • Compare fragmentation patterns with theoretical predictions

• Cross-reactivity Testing:

  SOCS Family Member	Sequence Homology with SOCS2	Expected Cross-reactivity	Distinguishing Features
  SOCS1	Low in N-terminal region	Minimal	Contains KIR domain absent in SOCS2
  SOCS3	Moderate in SH2 domain	Possible	Contains KIR domain absent in SOCS2
  CIS	High in SH2 domain	More likely	Different molecular weight (28 kDa)

The SOCS2-SH2 domain has unique structural features including specific interaction with both phosphorylated and non-phosphorylated peptides via different binding sites, which can be leveraged for selective detection .

How can SOCS2 antibodies be utilized to investigate the dual role of SOCS2 in both promoting and inhibiting cytokine signaling?

SOCS2 exhibits the intriguing capability to either positively or negatively regulate GH/cytokine signaling based on context . To investigate this dual functionality:

• Concentration-Dependent Effects:

  • Design dose-response experiments using increasing concentrations of SOCS2 expression

  • Monitor JAK-STAT signaling outputs using phospho-specific antibodies

  • Analyze SOCS2-GHR and SOCS2-JAK2 complex formation using co-immunoprecipitation with biotinylated SOCS2 antibodies

• Temporal Dynamics Analysis:

  • Perform time-course experiments after cytokine stimulation

  • Use biotinylated SOCS2 antibodies for ChIP-seq to identify SOCS2-dependent transcriptional changes

  • Implement proximity ligation assays to visualize SOCS2 interactions in situ

• Competitive Binding Studies:

  • Investigate SOCS2 interactions with other SOCS family members

  • Examine how SOCS2 potentially promotes signaling by displacing more potent negative regulators

  • Use SOCS2 antibodies in combination with other SOCS antibodies in sequential immunoprecipitation

This approach has revealed that SOCS2 can function as a molecular switch in signaling pathways, with its E3 ligase activity potentially targeting other SOCS proteins for degradation under specific conditions .

What methodological approaches can be used to investigate SOCS2's role in immune response regulation and bacterial infection responses?

SOCS2 has been implicated in regulating immune responses to bacterial infection, particularly mastitis in sheep . To investigate this function:

• Infection Models:

  • Establish in vitro infection systems using relevant bacterial pathogens

  • Monitor SOCS2 expression using biotinylated antibodies for flow cytometry

  • Analyze SOCS2 recruitment to relevant signaling complexes following pathogen recognition

• Cytokine Profiling:

  • Measure how SOCS2 modulates LPS and IFNγ responses

  • Use multiplexed cytokine assays to profile inflammatory mediators

  • Compare wild-type and SOCS2-deficient cells using SOCS2 antibodies to confirm knockout efficiency

• Genetic Approaches:

  • Utilize SOCS2R96C mouse model to assess immune responses

  • Implement CRISPR-Cas9 to generate cell lines with specific SOCS2 mutations

  • Conduct genetic association studies in disease cohorts combined with protein expression analysis

The research indicates that SOCS2's role in bacterial infection responses is likely related to its upregulation by LPS and IFNγ, and its inhibition of specific cytokine signaling pathways critical for immune regulation .

How might new structural insights into the SOCS2-SH2 domain inform the development of more specific research tools and therapeutic approaches?

Recent discoveries of non-canonical binding sites on the SOCS2-SH2 domain open exciting research avenues :

• Next-Generation Antibody Development:

  • Design antibodies specifically targeting the exosite region

  • Develop conformation-specific antibodies that recognize active vs. inactive SOCS2

  • Create domain-specific tools that differentially detect SOCS2 in various binding states

• Structural Biology Applications:

  • Implement hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map conformational changes

  • Use cryo-EM to resolve full SOCS2-GHR-JAK2 complexes

  • Apply computational modeling to predict binding interfaces and design specific inhibitors

• Therapeutic Implications:

  • Design peptide mimetics targeting the exosite for selective SOCS2 modulation

  • Develop small molecules that could selectively enhance or inhibit SOCS2 activity

  • Explore SOCS2-based biologics for growth disorders or inflammatory conditions

The model where SOCS2 can simultaneously engage multiple phosphorylated sites, potentially on both subunits of the dimerized GHR, provides a mechanistic framework for developing tools that can distinguish between different functional states of SOCS2 .

What experimental approaches can address contradictory findings regarding SOCS2's effect on JAK-STAT signaling across different cell types and contexts?

Contradictory findings regarding SOCS2's regulatory roles demand sophisticated experimental strategies:

• Cell-Type Specific Analysis:

  • Compare SOCS2 function across diverse cell lineages

  • Use tissue-specific inducible knockout models

  • Employ single-cell approaches combined with biotinylated SOCS2 antibodies for phenotyping

• Context-Dependent Signaling:

  • Establish defined experimental systems with controlled cytokine environments

  • Investigate SOCS2 function under varying concentrations of growth hormone and other cytokines

  • Analyze competitive interactions between different SOCS family members using quantitative proteomics

• Post-translational Regulation:

  • Examine how SOCS2 itself is regulated by phosphorylation or other modifications

  • Investigate protein half-life and turnover rates in different cellular contexts

  • Develop antibodies specific for modified forms of SOCS2