SOCS7 Antibody, HRP conjugated

What is the biological function of SOCS7 and why is it a significant research target?

SOCS7 functions as a substrate-recognition component of a cullin-5-RING E3 ubiquitin-protein ligase complex (ECS complex, also named CRL5 complex), which mediates the ubiquitination and subsequent proteasomal degradation of target proteins such as DAB1 and IRS1. It specifically recognizes and binds phosphorylated proteins via its SH2 domain, promoting their ubiquitination. The ECS(SOCS7) complex plays a crucial role in regulating reelin signaling by mediating the ubiquitination and degradation of phosphorylated DAB1 in the developing cerebral cortex, thereby influencing neuron positioning during cortical development. Additionally, SOCS7 functions in insulin signaling and glucose homeostasis through IRS1 ubiquitination and proteasomal degradation. It also inhibits prolactin, growth hormone, and leptin signaling by preventing STAT3 and STAT5 activation, sequestering them in the cytoplasm and reducing their DNA binding capacity .

Recent research has demonstrated that SOCS7 inhibits high-grade serous ovarian cancer (HGSOC) tumorigenesis through regulating the RNA-binding protein HuR and FOXM1, suggesting that SOCS7 could be a prospective biomarker for this cancer type .

What are the key considerations when selecting a SOCS7 antibody for HRP conjugation experiments?

When selecting a SOCS7 antibody for HRP conjugation experiments, researchers should consider:

• Antibody format: Choose antibodies specifically designed for conjugation, such as those in a "conjugation-ready format" that are carrier-free (BSA-free and azide-free), as these are optimized for labeling with enzymes like HRP .

• Antibody specificity: Verify that the antibody has been validated for specific recognition of SOCS7 protein with minimal cross-reactivity to other SOCS family members.

• Species reactivity: Ensure the antibody reacts with your species of interest. Some SOCS7 antibodies are specifically validated for human samples but may work with other species based on sequence homology .

• Application compatibility: Confirm the antibody has been validated for your intended applications (e.g., WB, IHC-P) when conjugated to HRP.

• Clonality: Consider whether a monoclonal antibody (like recombinant rabbit monoclonal) might be more suitable than polyclonal for consistent results in quantitative experiments.

How does the lyophilization process enhance HRP-antibody conjugation efficiency?

Lyophilization (freeze-drying) has been shown to significantly enhance the HRP-antibody conjugation process, resulting in conjugates with greater sensitivity compared to traditional methods. The modified procedure involves:

• Activation of HRP: Horseradish peroxidase is first activated using sodium meta-periodate to generate aldehyde groups by oxidation of carbohydrate moieties on the HRPO molecule.

• Lyophilization step: The activated form of HRPO is then lyophilized (freeze-dried) before being mixed with antibodies at a concentration of 1 mg/ml.

• Enhanced performance: This modified approach produces conjugates that demonstrate significantly higher sensitivity in immunoassays. In comparative studies, conjugates prepared with the lyophilization step worked effectively at dilutions as high as 1:5000, whereas conjugates prepared by the classical method required much lower dilutions (1:25) to achieve detectable signals .

The chemical modification of HRP during the conjugation procedure can be confirmed by UV spectroscopy, which typically shows a shift in absorption resulting in a small peak at 430 nm. The successful conjugation can be further verified using SDS-PAGE analysis .

This enhanced conjugation method appears to enable antibodies to bind more HRP molecules, significantly improving detection sensitivity in assays like ELISA.

What are the most effective methods to verify successful HRP conjugation to SOCS7 antibodies?

Verification of successful HRP conjugation to SOCS7 antibodies can be accomplished through several complementary methods:

• UV-Visible Spectroscopy: Successful conjugation typically results in a shift in the absorption spectrum, with a characteristic small peak at around 430 nm due to the modification of HRPO during the conjugation procedure .

• SDS-PAGE Analysis: This can confirm the increase in molecular weight of the antibody-HRP complex compared to the unconjugated antibody. Successful conjugation will show a band at a higher molecular weight corresponding to the antibody plus the HRP enzyme (approximately 44 kDa for HRP) .

• Functional Activity Testing: The most definitive verification comes from testing the enzymatic activity of the conjugate in an actual immunoassay:

  • Direct ELISA against purified SOCS7 protein

  • Western blot analysis using cell lysates known to express SOCS7

  • Compare signal intensity at various dilutions to commercial HRP-conjugated antibodies

• Enzymatic Activity Assay: Test the peroxidase activity of the conjugate using standard HRP substrates (TMB, DAB, or chemiluminescent substrates) in the absence of antigen binding to confirm the enzyme component remains active.

For optimal experimental design, researchers should perform both physical characterization (UV-Spec and SDS-PAGE) and functional testing to ensure both successful conjugation and retention of both antibody binding and enzymatic activities.

How can SOCS7 antibodies be optimally applied in ubiquitination assays?

SOCS7 antibodies play a crucial role in ubiquitination assays due to SOCS7's function in the ubiquitin-proteasome pathway. Here's a methodological approach based on published research:

• Sample Preparation:

  • Prepare cell lysates using lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris pH7.5, and 1 mM EDTA)

  • Supplement with protease inhibitor cocktail to prevent protein degradation

  • Include the proteasome inhibitor MG132 in treatments to preserve ubiquitinated proteins

• Immunoprecipitation Protocol:

  • Incubate lysates with anti-SOCS7 antibody (or antibody against the target protein of interest)

  • Use normal IgG as a negative control

  • Add Protein A/G PLUS-Agarose beads and incubate at 4°C for 2 hours

  • Wash the immunocomplex three times with lysis buffer

• Detection Methods:

  • Perform Western blot analysis with antibodies against SOCS7, the target protein (e.g., HuR), and ubiquitin

  • For effective ubiquitin detection, use anti-ubiquitin antibodies specifically validated for this application

• Controls and Validation:

  • Include samples treated with and without proteasome inhibitors

  • Compare wild-type cells with SOCS7-overexpressing and SOCS7-knockdown cells

  • Use MG132 treatment to rescue protein levels of suspected SOCS7 targets

This methodology has been successfully applied to demonstrate that SOCS7 mediates the ubiquitination of proteins like HuR in cancer cells and JAK2 in stem cells, providing insights into the molecular mechanisms of SOCS7 function .

What methodological approaches are recommended for studying SOCS7's role in JAK-STAT pathway regulation?

SOCS7 is known to inhibit cytokine signaling by preventing STAT3 and STAT5 activation. Here are recommended methodological approaches for investigating this regulatory mechanism:

• Cellular Models:

  • Establish SOCS7-knockdown and SOCS7-overexpression cell models using lentiviral transduction or other gene manipulation techniques

  • Choose relevant cell types based on your research focus (e.g., ovarian cancer cells for tumor studies, stem cells for differentiation studies)

• JAK2 Ubiquitination Assay:

  • Extract total protein from cells with or without SOCS7 peptide treatment

  • Immunoprecipitate with anti-JAK2 antibody using Protein A/G

  • Separate by SDS-PAGE and transfer to nitrocellulose membranes

  • Probe Western blots with anti-ubiquitin antibody to assess JAK2 ubiquitination levels

• JAK-STAT Pathway Analysis:

  • Examine JAK2 and STAT3/STAT5 phosphorylation status using phospho-specific antibodies

  • Assess nuclear translocation of STAT proteins using nuclear/cytoplasmic fractionation followed by Western blotting

  • Use co-immunoprecipitation to investigate protein interactions between SOCS7 and JAK/STAT components

• Functional Validation:

  • Measure downstream target gene expression using qRT-PCR

  • Assess cellular phenotypes relevant to JAK-STAT signaling (proliferation, differentiation)

  • Use STAT-responsive reporter assays to measure transcriptional activity

Research has demonstrated that SOCS7 peptide treatment promotes JAK2 ubiquitination, leading to inhibition of the JAK2-STAT3 pathway. This mechanism has been implicated in processes such as neuronal differentiation of adipose-derived mesenchymal stem cells .

How can SOCS7 antibodies be used to investigate cancer pathogenesis, particularly in ovarian cancer?

SOCS7 antibodies have proven valuable for investigating cancer pathogenesis, particularly in high-grade serous ovarian cancer (HGSOC). Here's a comprehensive methodological approach:

• Correlation with Clinical Outcomes:

  • Perform immunohistochemical analysis of SOCS7 expression in patient tumor samples

  • Correlate expression levels with clinicopathological characteristics (FIGO stage, grade) and survival outcomes

  • Use multivariate analysis to determine if SOCS7 is an independent prognostic factor

• Mechanistic Studies:

  • Create cellular models with modified SOCS7 expression:

    • SOCS7-knockdown using shRNA or siRNA

    • SOCS7-overexpression via lentiviral transduction

  • Assess effects on cell viability, colony formation, cell cycle progression, and apoptosis

  • Use in vivo xenograft models to confirm in vitro findings

• Protein-Protein Interaction Studies:

  • Employ co-immunoprecipitation (co-IP) with SOCS7 antibodies to identify interacting partners

  • Confirm interactions through reciprocal co-IP experiments

  • Use immunofluorescence co-localization studies to visualize SOCS7 and interacting proteins

• Ubiquitination Pathway Analysis:

  • Investigate SOCS7's role in mediating ubiquitination of target proteins

  • Use proteasome inhibitors (e.g., MG132) to confirm the involvement of proteasomal degradation

  • Perform ubiquitination assays to demonstrate direct effects on target proteins

Research has revealed that SOCS7 expression correlates positively with survival rates in HGSOC patients. Mechanistically, SOCS7 inhibits cancer cell viability by mediating the ubiquitination of HuR, an RNA-binding protein that promotes oncogenesis. This leads to reduced FOXM1 expression, a transcription factor involved in cell proliferation and cell cycle progression .

What are the optimal protocols for co-immunoprecipitation assays using SOCS7 antibodies?

Co-immunoprecipitation (co-IP) is a powerful technique for studying protein-protein interactions involving SOCS7. Here's an optimal protocol based on published research:

• Lysate Preparation:

  • Harvest cells and prepare lysates using an appropriate lysis buffer (e.g., 1% Triton X-100, 150 mM NaCl, 20 mM Tris pH7.5, 1 mM EDTA)

  • Supplement with protease inhibitor cocktail

  • Clarify lysates by centrifugation at 12,000 × g for 10 min at 4°C

• Immunoprecipitation:

  • Incubate clarified lysates with:

    • Anti-SOCS7 antibody to pull down SOCS7 and associated proteins

    • Antibody against suspected interacting partners (e.g., anti-HuR)

    • Normal IgG as a negative control

  • Add Protein A/G PLUS-Agarose beads and incubate at 4°C for 2 hours

  • Wash the immunocomplex three times with lysis buffer

• Detection and Analysis:

  • Perform SDS-PAGE and transfer to membranes

  • Immunoblot with antibodies against SOCS7 and potential interacting partners

  • For reciprocal confirmation, perform the co-IP in reverse (pull down with partner antibody, detect SOCS7)

• Controls and Validation:

  • Input control: Analysis of a small portion of the lysate before immunoprecipitation

  • IgG control: Immunoprecipitation with non-specific IgG

  • Validation by immunofluorescence co-localization studies

This approach has successfully demonstrated the interaction between SOCS7 and HuR in ovarian cancer cells, revealing a novel mechanism by which SOCS7 regulates HuR protein levels through ubiquitin-mediated proteasomal degradation .

What strategies can resolve high background issues when using HRP-conjugated SOCS7 antibodies?

High background is a common challenge when working with HRP-conjugated antibodies. Here are evidence-based strategies to minimize background and improve signal-to-noise ratio:

• Optimization of Antibody Dilution:

  • Perform titration experiments to determine the optimal antibody concentration

  • Start with higher dilutions (1:5000) for HRP-conjugated antibodies prepared using enhanced methods

  • Traditional conjugates may require much lower dilutions (1:25 to 1:100)

• Blocking Optimization:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Extend blocking time to ensure complete coverage of non-specific binding sites

  • Add 0.1-0.5% Tween-20 to blocking and washing buffers

• Washing Protocol Enhancement:

  • Increase the number of wash steps (minimum 3-5 washes)

  • Extend washing times (5-10 minutes per wash)

  • Use larger volumes of wash buffer

• Substrate Selection and Development:

  • Choose appropriate substrates based on sensitivity requirements

  • For Western blots or IHC, consider TMB or DAB with controlled development times

  • For enhanced sensitivity, use chemiluminescent substrates with optimized exposure times

• Sample-Specific Considerations:

  • Pre-absorb antibodies with tissue/cell powder from relevant negative samples

  • Consider species-specific secondary HRP conjugates if using primary antibodies from multiple species

  • For tissues with high endogenous peroxidase activity, use peroxidase quenching steps before antibody application

The lyophilization-enhanced conjugation method has been shown to significantly improve signal-to-noise ratio, allowing for much higher dilutions (1:5000) while maintaining specific signal detection .

How can contradictory results in SOCS7 signaling studies be reconciled through methodological improvements?

Contradictory results in SOCS7 signaling studies can arise from various methodological factors. Here are strategies to reconcile discrepancies:

• Standardization of Experimental Models:

  • Use well-characterized cell lines with defined SOCS7 expression levels

  • Create isogenic cell models with SOCS7 knockdown, knockout, or overexpression

  • Confirm SOCS7 expression status using multiple detection methods (qRT-PCR, Western blot, immunofluorescence)

• Comprehensive Pathway Analysis:

  • Examine multiple components of the signaling pathway simultaneously

  • Assess both protein levels and phosphorylation status of JAK-STAT pathway components

  • Investigate temporal dynamics of signaling changes following stimulation

• Context-Dependent Function Assessment:

  • SOCS7 may have different effects depending on cell type and environmental context

  • Compare results across multiple cell types relevant to your research question

  • Consider the influence of culture conditions, cell density, and growth factors

• Technical Validation Approaches:

  • Use multiple antibodies targeting different epitopes of SOCS7

  • Employ complementary techniques to confirm key findings (e.g., verify protein interactions using both co-IP and immunofluorescence co-localization)

  • Implement rigorous controls (positive, negative, isotype)

• Quantitative Analysis:

  • Use quantitative methods with statistical analysis

  • Perform dose-response and time-course experiments

  • Replicate experiments multiple times with biological replicates

Research has shown that SOCS7 functions differently in various contexts - it acts as a tumor suppressor in ovarian cancer by mediating HuR ubiquitination , while in stem cells it facilitates neuronal differentiation through JAK2 ubiquitination and STAT3 pathway inhibition . Recognizing these context-dependent roles is crucial for reconciling apparently contradictory findings.