SOCS2 Antibody, FITC conjugated

What is SOCS2 and what are its primary functions in cellular signaling?

SOCS2 (Suppressor of cytokine signaling 2) functions as a key negative regulator of growth hormone (GH) and insulin-like growth factor 1 (IGF-1) signaling pathways, which are associated with acromegaly and various cancers. It serves as a substrate-recognition component within the cullin-5-RING E3 ubiquitin-protein ligase complex (also called the ECS or CRL5 complex), mediating the ubiquitination and subsequent proteasomal degradation of target proteins, including growth hormone receptor (GHR) and erythropoietin receptor (EPOR) . SOCS2 specifically recognizes and binds phosphorylated proteins through its SH2 domain, which is characteristic of the SOCS family proteins . Research has demonstrated that SOCS2 plays a protective role in the liver by controlling pro-oxidative and inflammatory mechanisms, as evidenced in acetaminophen overdose models .

What is the molecular structure of SOCS2 and how does it relate to its function?

SOCS2 contains a central Src homology 2 (SH2) domain, which is crucial for recognizing and binding to phosphorylated tyrosine residues in target proteins. The SH2 domain consists of three β-strands flanked by two α-helices, forming two distinct pockets: a highly conserved phosphotyrosine binding pocket and a hydrophobic pocket that typically accommodates hydrophobic residues in the target protein . Recent structural studies have identified an exosite on the SOCS2-SH2 domain that, when bound to a non-phosphorylated peptide (F3), enhances the SH2 domain's affinity for canonical phosphorylated ligands . The F3 peptide binds as an α-helix on the opposite side of the SH2 domain from the phosphopeptide binding site, stabilizing the domain and resulting in slower dissociation of phosphorylated ligands, ultimately enhancing binding affinity and SOCS2 inhibition of GH signaling .

How does FITC conjugation affect SOCS2 antibody applications?

FITC (Fluorescein isothiocyanate) conjugation to SOCS2 antibodies provides direct fluorescent detection capabilities without requiring secondary antibodies, enhancing experimental efficiency. The conjugation allows for visualization of SOCS2 protein localization in cellular contexts using fluorescence microscopy, flow cytometry, and immunofluorescence assays . When using SOCS2 antibody with FITC conjugation, researchers should consider that the fluorophore has an excitation maximum at approximately 495 nm and an emission maximum around 519 nm, placing it in the green spectrum of visible light. Storage conditions for FITC-conjugated antibodies typically require -20°C to -80°C temperatures, with protection from repeated freeze-thaw cycles to maintain fluorescence intensity . It's important to note that photobleaching can occur with prolonged exposure to light, so samples should be protected during storage and handling.

What are the recommended applications for SOCS2 antibody, FITC conjugated?

SOCS2 antibody, FITC conjugated is primarily recommended for fluorescence-based detection methods including immunofluorescence microscopy, flow cytometry, and fluorescence-activated cell sorting (FACS). While the product information indicates Western blot compatibility with human and mouse samples , the FITC conjugation is particularly advantageous for applications requiring direct visualization of SOCS2 protein in cellular contexts . When working with fixed cells or tissues, researchers should optimize fixation and permeabilization protocols to preserve both antibody binding capacity and FITC fluorescence. For flow cytometry applications, typical working dilutions range from 1:50 to 1:200, though optimization for specific experimental conditions is recommended. When performing co-localization studies, researchers should select complementary fluorophores with minimal spectral overlap to avoid bleed-through artifacts.

How can SOCS2 antibody, FITC conjugated be utilized to investigate JAK-STAT signaling pathway dynamics?

To investigate JAK-STAT signaling dynamics using FITC-conjugated SOCS2 antibody, researchers should design time-course experiments following stimulation with relevant cytokines or growth factors. The experimental approach should include: (1) Treating cells with GH or other STAT-activating cytokines at defined time points (0, 5, 15, 30, 60, 120 minutes); (2) Fixing and permeabilizing cells to allow antibody access to intracellular SOCS2; (3) Staining with FITC-conjugated SOCS2 antibody alongside phospho-STAT5 antibodies with compatible fluorophores; and (4) Analyzing by flow cytometry or confocal microscopy to track SOCS2 induction relative to STAT activation . This approach can reveal the temporal relationship between STAT activation and SOCS2 upregulation, demonstrating the negative feedback loop. For quantitative assessment, mean fluorescence intensity measurements from flow cytometry can be plotted against time to generate kinetic profiles of SOCS2 expression following pathway activation. Additionally, co-immunoprecipitation experiments using the antibody can identify temporal association of SOCS2 with GHR and JAK2, providing insights into the mechanisms of signal termination .

What methodological approaches can be employed to study SOCS2-mediated protein degradation using the FITC-conjugated antibody?

To study SOCS2-mediated protein degradation, researchers can implement a multifaceted approach combining the FITC-conjugated SOCS2 antibody with proteasome inhibitors and ubiquitination analysis. The methodology should include: (1) Treating cells with proteasome inhibitors (e.g., MG132 at 10 μM for 4-6 hours) to accumulate ubiquitinated proteins; (2) Using the FITC-conjugated SOCS2 antibody for immunoprecipitation of SOCS2 complexes; (3) Analyzing co-precipitated proteins by Western blotting for known targets such as GHR or EPOR; and (4) Probing for ubiquitin to confirm target protein modification . For live-cell imaging of degradation dynamics, researchers can transfect cells with fluorescently-tagged substrate proteins (distinct from FITC wavelength) and track their co-localization with SOCS2-FITC signals over time using confocal microscopy. Pulse-chase experiments combining cycloheximide treatment with time-course analysis of substrate protein levels can quantify degradation rates in SOCS2-overexpressing versus SOCS2-knockout cells. Additionally, researchers can employ fluorescence resonance energy transfer (FRET) between FITC-labeled SOCS2 and appropriately labeled substrate proteins to monitor real-time interaction dynamics in living cells .

How can FITC-conjugated SOCS2 antibody be used to evaluate the newly discovered exosite interactions on the SH2 domain?

To investigate the exosite interactions on the SOCS2-SH2 domain using FITC-conjugated SOCS2 antibody, researchers should design competitive binding assays and structural stability experiments. The methodology should include: (1) Developing an in vitro fluorescence polarization assay using FITC-conjugated SOCS2 antibody to detect conformational changes upon F3 peptide binding to the exosite; (2) Performing thermal shift assays with the antibody in the presence and absence of F3 peptide to measure stabilization effects on the SH2 domain; (3) Conducting competitive binding experiments with varying concentrations of phosphotyrosine-containing peptides and F3 peptide to validate the enhancement effect quantitatively; and (4) Using the FITC-conjugated antibody in microscopy-based FRET assays with target proteins labeled with compatible acceptor fluorophores to visualize interaction dynamics in cellular contexts . For advanced structural analysis, researchers can combine antibody epitope mapping with hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions of SOCS2 that undergo conformational changes upon F3 binding. Additionally, site-directed mutagenesis of key residues in the exosite can be evaluated using the FITC-conjugated antibody to determine critical interaction points for the allosteric enhancement effect .

What are the implications of SOCS2's role in inflammatory responses and how can FITC-conjugated antibodies help investigate this function?

To investigate SOCS2's role in inflammatory responses, researchers can employ FITC-conjugated SOCS2 antibodies in multi-parameter flow cytometry and in vivo imaging studies. The experimental design should include: (1) Establishing inflammation models such as acetaminophen-induced liver injury in wild-type and SOCS2-knockout mice; (2) Analyzing immune cell populations by flow cytometry with FITC-conjugated SOCS2 antibody alongside markers for neutrophils, macrophages, and other inflammatory cells; (3) Performing intravital microscopy in transparent tissue windows using the fluorescent antibody to track SOCS2 expression dynamics during inflammation progression; and (4) Correlating SOCS2 expression levels with pro-inflammatory cytokine production and reactive oxygen species (ROS) generation . For mechanistic studies, researchers can isolate primary immune cells from inflamed tissues and use the FITC-conjugated antibody to sort SOCS2-high versus SOCS2-low populations for downstream functional analysis. Additionally, combining SOCS2 detection with phospho-flow cytometry for JAK-STAT pathway components can reveal the relationship between SOCS2 expression and inflammatory signal regulation in specific immune cell subtypes. Confocal microscopy of tissue sections using the FITC-labeled antibody alongside markers of tissue damage can provide spatial information about SOCS2's protective function in affected organs .

What are the optimal protocols for using SOCS2 antibody, FITC conjugated in flow cytometry experiments?

For optimal flow cytometry protocols using FITC-conjugated SOCS2 antibody, researchers should follow these methodological steps: (1) Harvest cells and fix with 2-4% paraformaldehyde for 15 minutes at room temperature; (2) Permeabilize cells using 0.1% Triton X-100 or saponin-based permeabilization buffer for 10 minutes; (3) Block with 5% normal serum corresponding to the antibody host species (rabbit) for 30 minutes; (4) Incubate with FITC-conjugated SOCS2 antibody at a 1:50 to 1:100 dilution in blocking buffer for 1 hour at room temperature or overnight at 4°C; (5) Wash three times with PBS containing 0.1% BSA; and (6) Analyze using appropriate flow cytometer settings for FITC detection (488 nm excitation laser, 530/30 nm bandpass filter) . For multi-parameter analysis, select compatible fluorophores with minimal spectral overlap with FITC. When performing intracellular staining, inclusion of protein transport inhibitors (like Brefeldin A or Monensin) during cell stimulation can enhance detection of transiently expressed proteins. For quantitative applications, include appropriate isotype controls (rabbit IgG-FITC) and consider using calibration beads to standardize fluorescence measurements across experiments.

How can SOCS2 antibody, FITC conjugated be used in immunofluorescence microscopy studies?

For immunofluorescence microscopy using FITC-conjugated SOCS2 antibody, researchers should implement the following protocol: (1) Culture cells on glass coverslips or prepare tissue sections at 5-10 μm thickness; (2) Fix samples with 4% paraformaldehyde for 15 minutes, followed by permeabilization with 0.2% Triton X-100 for 10 minutes; (3) Block with 5% normal goat serum in PBS containing 0.1% BSA for 1 hour at room temperature; (4) Incubate with FITC-conjugated SOCS2 antibody at 1:100 dilution in blocking buffer overnight at 4°C in a humidified chamber protected from light; (5) Wash three times with PBS for 5 minutes each; (6) Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes; and (7) Mount using anti-fade mounting medium . For co-localization studies, combine with antibodies against GHR, JAK2, or STAT5 labeled with spectrally distinct fluorophores. When examining subcellular localization, include markers for relevant organelles such as nuclear envelope or cytoskeletal components. To minimize photobleaching during imaging, use appropriate anti-fade reagents and optimize acquisition parameters to use the minimum excitation intensity needed for adequate signal detection. For quantitative analysis of fluorescence intensity, include calibration standards and use software that can perform normalized measurements across experimental conditions.

What is the recommended protocol for cell-penetrating SOCS2 protein delivery and tracking using FITC-conjugated antibodies?

For cell-penetrating SOCS2 protein delivery and tracking, researchers should follow this methodological approach: (1) Produce recombinant SOCS2 protein containing a membrane-permeable peptide sequence as described in previous studies; (2) Apply the cell-penetrating SOCS2 protein to target cells at concentrations of 0.5-2 μM for 1-4 hours in serum-free medium; (3) Wash cells extensively to remove extracellular protein; (4) Fix cells with 4% paraformaldehyde for 15 minutes, then permeabilize with 0.1% Triton X-100; (5) Block with 5% BSA for 30 minutes; (6) Incubate with FITC-conjugated SOCS2 antibody (1:100 dilution) for 1-2 hours to detect internalized SOCS2 protein; and (7) Analyze by confocal microscopy or flow cytometry . For live tracking experiments, consider dual-labeling strategies where the cell-penetrating SOCS2 protein is directly labeled with a fluorophore spectrally distinct from FITC, allowing differentiation between exogenous and endogenous SOCS2. Time-course experiments should include multiple timepoints (0.5, 1, 2, 4, 8, 24 hours) to track protein internalization, intracellular distribution, and eventual degradation. For functional validation, combine with phospho-specific antibodies against STAT5 to demonstrate inhibition of GH-STAT5 signaling in parallel with protein internalization .

How can SOCS2 antibody, FITC conjugated be used to study the ubiquitination pathway in different cell types?

To study SOCS2-mediated ubiquitination using FITC-conjugated antibody across different cell types, researchers should implement the following protocol: (1) Establish cell models representing diverse tissues (e.g., hepatocytes, immune cells, cancer cell lines) with varying levels of SOCS2 expression; (2) Treat cells with proteasome inhibitors (MG132 at 10 μM for 4-6 hours) to accumulate ubiquitinated proteins; (3) Perform dual immunofluorescence staining with FITC-conjugated SOCS2 antibody and anti-ubiquitin antibodies with compatible fluorophores; (4) Analyze co-localization by confocal microscopy with quantitative co-localization analysis; and (5) Validate findings with biochemical ubiquitination assays including immunoprecipitation and Western blotting . For tissue-specific analysis, prepare sections from different organs and perform similar staining to identify cell types with high SOCS2-ubiquitin co-localization. Flow cytometry can be used for quantitative assessment across multiple cell types simultaneously by staining with FITC-conjugated SOCS2 antibody, cell-type specific markers, and ubiquitin antibodies. To examine the substrate specificity across cell types, combine with antibodies against known SOCS2 targets (GHR, EPOR) and assess their co-localization with SOCS2 and ubiquitin signals in different cellular contexts .

What are common pitfalls when using FITC-conjugated antibodies and how can they be addressed?

Common pitfalls when using FITC-conjugated SOCS2 antibodies include: (1) Photobleaching, which can be minimized by protecting samples from light exposure during storage and processing, using anti-fade mounting media, and optimizing microscope settings to use minimal excitation intensity; (2) Autofluorescence, especially in tissue samples containing lipofuscin or elastin, which can be reduced by using Sudan Black B (0.1-0.3% in 70% ethanol) treatment for 10 minutes after antibody incubation; (3) Non-specific binding, which can be addressed by optimizing blocking conditions (testing various blockers like BSA, normal serum, or commercial blocking reagents) and including appropriate isotype controls; and (4) Low signal-to-noise ratio, which can be improved by adjusting antibody concentration, incubation time, and temperature . When working with fixed tissues, aldehyde-induced autofluorescence can be quenched using sodium borohydride (1 mg/ml in PBS) for 10 minutes before blocking. For flow cytometry applications, compensation is critical when multiplexing with other fluorophores, and single-color controls should always be included. pH sensitivity of FITC (optimal at pH 8.0) should be considered when selecting buffers, as acidic conditions can significantly reduce fluorescence intensity.

How can researchers validate the specificity of SOCS2 antibody, FITC conjugated in their experimental systems?

To validate SOCS2 antibody specificity, researchers should implement multiple complementary approaches: (1) Perform parallel staining in SOCS2 knockout or knockdown cells alongside wild-type controls to confirm signal reduction; (2) Conduct pre-absorption controls by incubating the antibody with excess recombinant SOCS2 protein (1-5 μg/ml) prior to staining; (3) Compare staining patterns with alternative SOCS2 antibodies recognizing different epitopes; (4) Correlate protein detection with mRNA expression through parallel RT-PCR or in situ hybridization; and (5) Validate subcellular localization against published reports of SOCS2 distribution . For functional validation, researchers can stimulate cells with growth hormone, which should induce SOCS2 expression as part of the negative feedback loop, resulting in increased FITC signal intensity. Western blot validation using the same antibody (if compatible) or alternative SOCS2 antibodies should show bands at the expected molecular weight (~22 kDa). When possible, mass spectrometry analysis of immunoprecipitated proteins can provide definitive confirmation of antibody specificity and identify potential cross-reactive proteins.

What considerations should be made when designing experiments to study SOCS2 interactions with its binding partners?

When designing experiments to study SOCS2 interactions, researchers should consider: (1) The dynamic and often transient nature of SOCS2 interactions with phosphorylated proteins, which may require crosslinking approaches (using 1-2% formaldehyde for 10 minutes) to stabilize complexes before immunoprecipitation; (2) The requirement for tyrosine phosphorylation of binding partners, necessitating treatment with appropriate cytokines (e.g., GH at 500 ng/ml for 10-15 minutes) and phosphatase inhibitors (sodium orthovanadate at 1 mM) during cell lysis; (3) The potential competition between the antibody and binding partners for overlapping epitopes, which may require epitope mapping or alternative antibodies; and (4) The impact of the FITC conjugation on binding site accessibility, which should be assessed through parallel experiments with unconjugated antibodies . For studying the recently discovered exosite interactions, include controls with and without F3 peptide to assess enhancement effects on binding. When investigating E3 ligase complex formation, consider the multiprotein nature of the complex (including Cullin5, Rbx2, and Elongins B/C) and include detection methods for these components alongside SOCS2. Proximity ligation assays (PLA) combining the FITC-conjugated SOCS2 antibody with antibodies against potential binding partners can provide sensitive detection of protein-protein interactions in situ with spatial resolution.

How can researchers optimize detection sensitivity for low-abundance SOCS2 protein in different tissue samples?

To optimize detection of low-abundance SOCS2 protein in tissues, researchers should implement these methodological enhancements: (1) Use antigen retrieval techniques optimized for phospho-proteins, such as pressure cooking in citrate buffer (pH 6.0) for 15 minutes, or Tris-EDTA buffer (pH 9.0) for paraffin sections; (2) Employ signal amplification systems like tyramide signal amplification (TSA), which can increase sensitivity 10-100 fold by depositing additional fluorophores at the site of antibody binding; (3) Optimize fixation protocols to preserve SOCS2 epitopes, testing different fixatives (paraformaldehyde, methanol, or acetone) and durations; and (4) Increase antibody concentration (up to 1:25 dilution) and incubation time (overnight at 4°C) while maintaining specificity through appropriate controls . For tissues with high autofluorescence, consider alternative detection systems that use fluorophores in different spectral ranges (away from green autofluorescence), or implement specialized imaging techniques like spectral unmixing on confocal microscopes capable of separating FITC signal from autofluorescence. Additionally, pre-enrichment techniques such as laser capture microdissection of regions with expected SOCS2 expression followed by immunoblotting can increase detection sensitivity in heterogeneous tissues.

How can SOCS2 antibody, FITC conjugated contribute to cancer research and potential therapeutic developments?

SOCS2 antibody, FITC conjugated can advance cancer research through multiple approaches: (1) Profiling SOCS2 expression across tumor types and correlating with clinical outcomes using tissue microarrays and quantitative microscopy; (2) Monitoring dynamic changes in SOCS2 levels during treatment with JAK-STAT pathway inhibitors using flow cytometry of tumor samples; (3) Investigating the potential of cell-penetrating SOCS2 proteins as therapeutic agents by tracking their delivery to and effects on cancer cells; and (4) Developing screening assays for compounds that enhance SOCS2 binding to oncogenic targets using fluorescence-based interaction assays . The recent discovery of an exosite on the SOCS2-SH2 domain that enhances binding affinity opens new avenues for therapeutic development targeting this allosteric site. Researchers can use the FITC-conjugated antibody to screen for compounds binding this exosite and enhancing SOCS2's tumor-suppressive functions . Additionally, the antibody can be employed in high-content screening workflows to identify novel regulators of SOCS2 stability or activity that might have therapeutic potential. For in vivo applications, the FITC-conjugated antibody could potentially be used for intraoperative visualization of tumors with aberrant SOCS2 expression following direct injection into accessible tumors.

What are the implications of recent SOCS2 structural discoveries for developing covalent inhibitors and how can FITC-conjugated antibodies assist in their evaluation?

Recent structural discoveries, particularly the identification of Cys111 as a target for covalent modification in the SOCS2-SH2 domain, provide new opportunities for inhibitor development that can be evaluated using FITC-conjugated SOCS2 antibodies . The methodological approach should include: (1) Developing competitive binding assays where potential covalent inhibitors compete with the FITC-conjugated antibody for binding to SOCS2, with displacement indicating successful target engagement; (2) Creating cellular thermal shift assays (CETSA) combining the thermal stability measurement with FITC-antibody detection to assess inhibitor binding in intact cells; (3) Performing microscopy-based co-localization studies between FITC-labeled SOCS2 and fluorescently-labeled inhibitors to visualize target engagement in situ; and (4) Developing high-throughput screening assays using flow cytometry with the FITC-conjugated antibody to rapidly evaluate libraries of potential inhibitors . For validation of the prodrug strategy described with pivaloyloxymethyl (POM) protecting groups, the FITC-conjugated antibody can be used to track changes in SOCS2 conformation or complex formation following prodrug unmasking in cellular environments. Researchers can also combine the antibody with cellular proteasome sensors to correlate inhibitor binding with changes in SOCS2-mediated protein degradation activity.

How can SOCS2 antibody, FITC conjugated be utilized in studying the role of SOCS2 in inflammatory and autoimmune conditions?

To investigate SOCS2's role in inflammatory and autoimmune conditions, researchers can apply FITC-conjugated SOCS2 antibodies in several methodological approaches: (1) Performing multi-parameter flow cytometry on immune cells from patients with autoimmune diseases to correlate SOCS2 expression with disease activity and inflammatory markers; (2) Conducting immunofluorescence microscopy on affected tissues to examine spatial relationships between SOCS2-expressing cells and inflammatory infiltrates; (3) Implementing in vitro stimulation assays with disease-relevant cytokines to track dynamic changes in SOCS2 expression across immune cell subsets; and (4) Developing ex vivo models where patient-derived cells are treated with cell-penetrating SOCS2 proteins and monitored for changes in inflammatory responses . The protective role of SOCS2 in liver inflammation models suggests potential applications in other inflammatory conditions. Researchers can use the FITC-conjugated antibody to track SOCS2 expression during disease progression in animal models of rheumatoid arthritis, inflammatory bowel disease, or multiple sclerosis. For mechanistic studies, combining SOCS2 detection with markers of oxidative stress and inflammatory signaling pathways can reveal how SOCS2 regulates the balance between protective and pathogenic immune responses in different tissue microenvironments .

What future technological developments might enhance the application of SOCS2 antibody, FITC conjugated in systems biology approaches?

Future technological developments that could enhance SOCS2 antibody applications include: (1) Integration with single-cell multi-omics platforms, combining FITC-based SOCS2 protein detection with transcriptomics or proteomics analysis of the same cells to create comprehensive molecular profiles; (2) Implementation in advanced microscopy techniques such as super-resolution microscopy (STORM, PALM) to visualize SOCS2 distribution at nanometer resolution within subcellular compartments; (3) Adaptation for organ-on-chip or organoid systems to monitor SOCS2 dynamics in physiologically relevant 3D tissue contexts; and (4) Development of biosensor systems combining the antibody with FRET-based reporters to enable real-time monitoring of SOCS2-target interactions in living cells . Emerging technologies like mass cytometry (CyTOF) could incorporate metal-tagged versions of SOCS2 antibodies to enable highly multiplexed profiling of signaling networks across heterogeneous cell populations. For in vivo applications, development of near-infrared fluorophore-conjugated variants could enable non-invasive imaging of SOCS2 expression in small animal models using appropriate reporter systems. Additionally, integration with artificial intelligence-based image analysis could enhance quantitative assessment of complex SOCS2 distribution patterns across tissues and experimental conditions.