SOCS2 Antibody, HRP conjugated

What is SOCS2 and why is it important in signaling research?

SOCS2 is a critical negative regulator of growth hormone (GH) and insulin-like growth factor 1 (IGF-1) signaling pathways. It contributes to several biological processes including metabolism, bone formation, neuronal development, cancer progression, and infection response . Research indicates SOCS2 exerts its regulatory function by binding to tyrosine-phosphorylated GH and IGF-1 receptors through its SH2 domain, effectively suppressing downstream signaling . Additionally, SOCS2 possesses ubiquitin ligase activity through its SOCS box, which binds to Elongin B and C, promoting the degradation of receptors and other SOCS family members .

Methodologically, when investigating SOCS2 function, researchers should consider:

• Examining both positive and negative regulatory effects on GH/cytokine signaling

• Monitoring changes in receptor levels through Western blotting

• Assessing ubiquitination status of target proteins

• Comparing wild-type and SOCS2-null models to understand phenotypic differences

What are the optimal conditions for Western blotting using SOCS2 antibody with HRP detection?

For optimal Western blotting results with SOCS2 antibody:

• Sample preparation: Use RIPA buffer supplemented with protease and phosphatase inhibitors.

• Gel electrophoresis: Load 20-30 μg of total protein per lane on 10-12% SDS-PAGE gels (SOCS2 has a molecular weight of approximately 22 kDa) .

• Transfer: Use PVDF membrane for optimal protein binding.

• Blocking: Block with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Primary antibody: Incubate with SOCS2 antibody at 1:1000 dilution overnight at 4°C .

• Secondary antibody: Apply HRP-conjugated goat-anti-rabbit or appropriate secondary antibody at 1:1000 dilution for 1 hour at room temperature .

• Detection: Use chemiluminescent substrate compatible with HRP for optimal signal detection.

Note: SOCS2 protein levels may be low in certain tissues under basal conditions, and expression is typically induced upon cytokine treatment , which should be considered when designing experiments.

How can I troubleshoot weak or no signal when detecting SOCS2?

When encountering weak or absent SOCS2 signal during immunodetection:

• Verify antibody specificity: Ensure your SOCS2 antibody has been validated for your specific application and species. Confirm the antibody recognizes endogenous levels of SOCS2 .

• Optimize protein extraction:

  • Use fresh tissue/cells and maintain cold temperatures during extraction

  • Include proteasome inhibitors (SOCS2 is rapidly degraded as part of its regulatory function)

  • Consider phosphatase inhibitors if examining phosphorylation-dependent interactions

• Enhance signal detection:

  • Increase primary antibody concentration or incubation time

  • Use high-sensitivity chemiluminescent substrates optimized for HRP

  • Consider signal amplification techniques like tyramide signal amplification

• Induce SOCS2 expression:

  • SOCS2 is typically induced by cytokine treatment

  • Consider pre-treating samples with GH, IL-6, or other relevant cytokines to upregulate SOCS2 expression

• Check technical parameters:

  • Ensure appropriate secondary antibody (species compatibility)

  • Verify HRP conjugate activity

  • Examine transfer efficiency using reversible protein staining

How can I assess SOCS2-mediated ubiquitination of GHR in experimental models?

To effectively investigate SOCS2-mediated ubiquitination of growth hormone receptor (GHR):

• Co-immunoprecipitation approach:

  • Immunoprecipitate using anti-ubiquitin antibody, then probe for GHR in Western blot

  • Alternatively, immunoprecipitate GHR and probe for ubiquitin

  • Compare ubiquitination patterns between wild-type and SOCS2-null models, as studies have shown markedly decreased GHR ubiquitination in SOCS2-null mice

• In vitro ubiquitination assay:

  • Reconstitute the ubiquitination reaction using purified components (E1, E2, SOCS2, ubiquitin, and GHR)

  • Detect ubiquitinated species using anti-ubiquitin antibodies

  • Quantify the degree of ubiquitination using densitometric analysis

• Cell-based ubiquitination assays:

  • Express tagged versions of SOCS2 and GHR in cell culture

  • Treat with proteasome inhibitors to prevent degradation of ubiquitinated species

  • Analyze changes in GHR levels using Western blotting (SOCS2 overexpression has been shown to decrease GHR protein levels by approximately 30%)

• Mutational analysis:

  • Generate SOCS box mutants of SOCS2 that cannot interact with the ubiquitination machinery

  • Compare effects on GHR stability and signaling pathway activation

This methodological approach provides comprehensive insights into the ubiquitin-dependent regulation of GH signaling by SOCS2.

What are the key considerations when investigating SOCS2 in cancer research using immunodetection methods?

When investigating SOCS2 in cancer contexts:

• Expression pattern analysis:

  • Multiple studies have shown altered SOCS2 expression in various cancers

  • Low expression of SOCS2 has been associated with poor clinical prognosis in non-small cell lung cancer (NSCLC) by affecting epithelial-mesenchymal transition (EMT) and causing drug resistance

  • Compare SOCS2 expression between tumor and adjacent normal tissues using immunohistochemistry or Western blotting

• Technical considerations:

  • Use tissue microarrays to analyze multiple patient samples simultaneously

  • Include positive and negative controls for accurate interpretation

  • Consider subcellular localization of SOCS2 (nuclear vs. cytoplasmic)

• Correlation with clinical data:

  • Establish associations between SOCS2 expression levels and patient survival, tumor stage, or treatment response

  • Analyze TCGA and GEO database information on SOCS2 expression across cancer types

• Pathway analysis:

  • Examine downstream effectors of GH/IGF-1 signaling in relation to SOCS2 expression

  • Investigate JAK-STAT pathway activation status

  • Analyze gene expression patterns through approaches like GSEA (Gene Set Enrichment Analysis)

• Drug resistance investigations:

  • Examine the relationship between SOCS2 levels and response to targeted therapies

  • Consider SOCS2 as a potential biomarker for treatment selection

How can I design experiments to distinguish between SOCS2's effects on GH signaling versus other cytokine pathways?

Designing experiments to disambiguate SOCS2's effects requires a systematic approach:

• Pathway-specific stimulation:

  • Selectively stimulate cells with GH versus other cytokines (IL-6, IFN-γ)

  • Monitor SOCS2 induction kinetics across different stimuli

  • Compare STAT phosphorylation patterns (STAT5 for GH, STAT1/3 for other cytokines)

• Receptor-specific analysis:

  • Examine SOCS2 binding to GHR versus other cytokine receptors using co-immunoprecipitation

  • Quantify receptor degradation rates in the presence/absence of SOCS2

  • Generate receptor chimeras to identify specific domains mediating SOCS2 interaction

• Downstream target profiling:

  • Perform transcriptomic analysis after pathway-specific stimulation in SOCS2-expressing versus SOCS2-deficient cells

  • Identify GH-specific versus cytokine-specific gene signatures

  • Use Ingenuity™ pathway analysis software to visualize activated/inhibited pathways

• Tissue-specific investigations:

  • Compare SOCS2 effects in liver (primary GH-responsive tissue) versus immune cells (cytokine-responsive)

  • Use conditional knockout models to eliminate SOCS2 in specific tissues

  • Assess physiological outcomes (growth versus immune response)

Experimental results indicate that SOCS2 knockout mice exhibit significantly greater hepatocyte proliferation when treated with GH compared to wild-type mice, suggesting a state of GH hyper-responsiveness in the absence of SOCS2 .

What controls should be included when using SOCS2 antibody for immunodetection?

A robust experimental design for SOCS2 antibody applications should include:

• Positive controls:

  • Cell lines known to express SOCS2 (especially after cytokine stimulation)

  • Recombinant SOCS2 protein

  • Cells transfected with SOCS2 expression vector

• Negative controls:

  • SOCS2 knockout/knockdown cells or tissues

  • Isotype control antibodies

  • Pre-adsorption of antibody with immunizing peptide

• Loading controls:

  • Housekeeping proteins (β-actin, GAPDH) for Western blotting

  • Total protein stains (Ponceau S) for membrane verification

• Specificity controls:

  • Detection of SOCS2 at the correct molecular weight (22 kDa)

  • Cross-reactivity assessment with other SOCS family members

  • Secondary antibody-only controls to assess non-specific binding

• Signal validation:

  • Verification of SOCS2 induction in response to known stimuli (e.g., GH, cytokines)

  • Correlation of protein levels with mRNA expression

  • Comparison of results using multiple detection methods

How can I optimize SOCS2 detection in liver regeneration studies?

For SOCS2 detection during liver regeneration research:

• Time-course considerations:

  • SOCS2 is induced early after liver injury to regulate hepatocyte proliferation

  • Design sampling intervals that capture early post-injury timepoints (6h, 24h, 36h, 48h)

  • Compare regenerative versus quiescent liver tissue

• Sample preparation:

  • Use fresh liver tissue or flash-freeze immediately after collection

  • Section preparation for immunohistochemistry should maintain tissue architecture

  • For Western blotting, prepare protein extracts with phosphatase inhibitors to preserve signaling status

• Detection strategy:

  • Combine protein detection (Western blot) with localization studies (immunohistochemistry)

  • Consider dual staining with proliferation markers (Ki67, PCNA)

  • Monitor both SOCS2 and GHR levels simultaneously

• Functional correlation:

  • Correlate SOCS2 expression with hepatocyte proliferation rates

  • Examine ubiquitination status of GHR at different regeneration timepoints

  • Analyze downstream pathway activation (particularly STAT1/3/5)

Research has demonstrated that after partial hepatectomy, GHR levels are higher in SOCS2-null mice compared to wild-type at 6 hours post-surgery, correlating with increased hepatocyte proliferation .

How should I interpret apparent contradictions in SOCS2 signaling effects?

SOCS2 exhibits seemingly contradictory effects across different experimental systems. To interpret these effectively:

Studies in mice have revealed that SOCS2 deficiency leads to significantly larger body size than normal littermates, supporting its inhibitory role in growth regulation .

What bioinformatic approaches can enhance SOCS2 research when combined with antibody-based detection?

Integrative bioinformatic approaches for SOCS2 research include:

• Expression correlation analysis:

  • Analyze SOCS2 expression patterns across public databases (TCGA, GEO)

  • Identify co-expressed genes that may function in common pathways

  • Correlate expression with clinical outcomes and disease progression

• Gene Set Enrichment Analysis (GSEA):

  • Use GSEA to identify biological pathways associated with SOCS2 expression levels

  • Compare gene signatures between SOCS2-high and SOCS2-low samples

  • Integrate with experimental data from antibody-based detection

• Network analysis:

  • Build protein-protein interaction networks centered on SOCS2

  • Identify key hub proteins that may be critical for SOCS2 function

  • Validate computational predictions using co-immunoprecipitation

• Mutation and variation analysis:

  • Examine tumor mutation burden in relation to SOCS2 expression

  • Analyze how genetic variations affect SOCS2 function and expression

  • Correlate mutation status with antibody-detected protein levels

• Multi-omics integration:

  • Combine proteomics, transcriptomics, and epigenomics data

  • Use machine learning to predict SOCS2 function in different contexts

  • Validate computational predictions experimentally

This approach has successfully identified differential activation of the GH signaling pathway in SOCS2-null mice during liver regeneration, as demonstrated by transcriptomic analysis .

How can I differentiate between SOCS2's direct effects and secondary compensatory mechanisms in experimental models?

To distinguish primary from secondary effects:

• Acute versus chronic models:

  • Use inducible expression/deletion systems (tetracycline-regulated, Cre-ERT2)

  • Compare immediate effects (minutes to hours) with long-term adaptation (days to weeks)

  • Monitor dynamic changes in signaling pathway components

• Rescue experiments:

  • Reintroduce wild-type SOCS2 in knockout models

  • Use domain mutants to identify specific functions (SH2 mutants, SOCS box mutants)

  • Examine which phenotypes are directly reversible

• Direct target identification:

  • Perform SOCS2 immunoprecipitation followed by mass spectrometry

  • Use proximity labeling approaches (BioID, TurboID) to identify nearby proteins

  • Validate direct interactions with co-immunoprecipitation

• Pathway inhibitor approach:

  • Block potential compensatory pathways pharmacologically

  • Examine if SOCS2 effects persist when secondary pathways are inhibited

  • Use combination of genetic and pharmacological approaches

• Temporal profiling:

  • Perform time-course experiments after SOCS2 manipulation

  • Identify immediate versus delayed gene expression changes

  • Establish causality using network modeling approaches

This methodology has revealed that SOCS2 directly controls liver GHR levels through ubiquitination, resulting in immediate changes to receptor levels within 6 hours of hepatectomy .

How can cell-penetrating SOCS2 proteins be developed and validated using immunodetection methods?

Cell-penetrating SOCS2 has therapeutic potential, particularly for conditions involving dysregulated GH/IGF-1 signaling:

• Design and production strategy:

  • Engineer recombinant SOCS2 containing membrane-permeable peptide sequences

  • Express in bacterial systems (E. coli) for high yield

  • Purify using affinity chromatography with appropriate tags

• Validation approaches:

  • Confirm protein delivery into target cells using immunofluorescence with SOCS2 antibodies

  • Quantify intracellular concentration using Western blotting with standard curves

  • Track cellular uptake kinetics using time-course experiments

• Functional assessment:

  • Monitor inhibition of cell growth in cancer cell lines

  • Examine downstream signaling effects (STAT5 phosphorylation)

  • Confirm interaction with target receptors (GHR) using co-immunoprecipitation

• Target validation:

  • Demonstrate downregulation of GH-STAT5 signaling target genes

  • Compare effects with conventional SOCS2 overexpression

  • Assess specificity by examining effects on related signaling pathways

Research has demonstrated that cell-penetrating SOCS2 proteins effectively enter cancer cell lines and inhibit cell growth by suppressing JAK-STAT5 signaling, suggesting therapeutic potential for conditions like acromegaly and certain cancers .

What considerations are important when examining SOCS2 in cancer and metabolism intersection research?

The SOCS2-mediated connection between metabolism and cancer requires specialized approaches:

• Metabolic phenotyping:

  • Examine glucose metabolism in SOCS2-modified cancer models

  • Assess lipid metabolism alterations in relation to SOCS2 expression

  • Monitor insulin sensitivity and GH responsiveness

• Cancer progression markers:

  • Correlate SOCS2 levels with epithelial-mesenchymal transition (EMT) markers

  • Examine drug resistance patterns in relation to SOCS2 expression

  • Assess tumor growth rates and metastatic potential

• Signaling node analysis:

  • Examine how SOCS2 modulates both metabolic and proliferative signaling

  • Investigate crosstalk between insulin/IGF-1 and inflammatory pathways

  • Assess how metabolic state influences SOCS2 function in cancer cells

• Translational considerations:

  • Stratify patient samples based on metabolic parameters and SOCS2 expression

  • Develop combination therapy approaches targeting both metabolic and oncogenic pathways

  • Explore SOCS2 as a biomarker for metabolically targeted cancer therapies

Research has shown that low SOCS2 expression correlates with poor clinical prognosis in NSCLC, potentially through effects on epithelial-mesenchymal transition and drug resistance mechanisms .

How can SOCS2 antibodies be used to study the temporal dynamics of signaling pathway regulation?

To investigate temporal regulation by SOCS2:

• Pulse-chase experimental design:

  • Stimulate cells with GH or cytokines for defined periods

  • Track SOCS2 induction, peak expression, and decay

  • Correlate with receptor levels and downstream signaling activation/deactivation

• Live-cell imaging approaches:

  • Generate fluorescently tagged SOCS2 constructs

  • Observe real-time recruitment to activated receptors

  • Correlate with signaling reporter systems (e.g., STAT nuclear translocation)

• Sequential immunoprecipitation:

  • Track dynamic protein complexes forming around SOCS2 at different timepoints

  • Identify temporal order of recruitment of ubiquitination machinery

  • Correlate with functional outcomes (receptor degradation, signal termination)

• Computational modeling:

  • Develop mathematical models of SOCS2-mediated feedback

  • Incorporate experimental data to refine model parameters

  • Predict system behavior under different conditions

• Multi-parameter flow cytometry:

  • Simultaneously assess SOCS2 levels, receptor abundance, and pathway activation

  • Analyze at single-cell resolution to capture population heterogeneity

  • Track changes over multiple timepoints after stimulation

This approach has demonstrated that SOCS2 is dynamically regulated during liver regeneration, with expression patterns that correlate with specific phases of the regenerative process .