SOS2 Antibody, HRP conjugated

What is SOS2 and what is its role in cellular signaling?

SOS2 is a 160-170 kDa protein that serves as a mediator of guanine nucleotide phosphate exchange in the Ras signaling pathway. In resting cells, cytoplasmic SOS2 forms a heterodimer with Grb2 and can also form a heterotrimer with Eps8 and E3b1. Following receptor tyrosine kinase (RTK) activation, the SOS2-Grb2 heterodimer is recruited to the cell membrane where it contacts GDP-bound Ras, facilitating a GTP-for-GDP exchange that activates Ras. Human SOS2 is 1332 amino acids in length with specific functional domains including a histone fold (amino acids 97-169), PH domain (amino acids 439-546), REM domain (amino acids 595-739) that interacts with Ras, and a proline-rich region (amino acids 1126-1242) that binds to Grb2 . SOS2 activity parallels that of SOS1, though SOS2 binds Grb2 with higher affinity but shows less biological activity due to a shorter half-life .

How does SOS2 differ from SOS1 in experimental systems?

While SOS1 and SOS2 share functional similarities in Ras activation, they exhibit important differences that researchers should consider when selecting antibodies and designing experiments:

These differences become particularly relevant when using SOS2 antibodies to study comparative roles of SOS proteins in signal transduction or when investigating compensatory mechanisms following genetic manipulation of either protein .

What applications are SOS2 antibodies typically used for?

SOS2 antibodies are successfully employed in multiple research applications, with varying optimization requirements:

• Western Blotting: Both polyclonal and monoclonal antibodies against SOS2 have demonstrated efficacy in western blot applications with expected band sizes of approximately 153-170 kDa . When using HRP-conjugated detection systems, researchers typically observe strong signals with short exposure times (30-75 seconds) using chemiluminescence .

• Immunoprecipitation: Rabbit polyclonal antibodies such as ab85831 have been validated for immunoprecipitation of SOS2 from whole cell lysates, typically using 3μg antibody per mg of lysate .

• Immunocytochemistry: SOS2 antibodies can be applied for cellular localization studies, particularly to investigate translocation events following receptor activation .

What are the critical criteria for selecting an appropriate SOS2 antibody?

When selecting an SOS2 antibody for research applications, consider these methodological factors:

• Epitope Location: Antibodies targeting different regions of SOS2 may yield varying results. For instance, some commercial antibodies target synthetic peptides within the C-terminal region (after amino acid 1250), while others may target functional domains . Epitope location can significantly impact antibody performance in applications where protein conformation is altered (denatured vs. native conditions).

• Species Reactivity: Verify cross-reactivity with your model system. Many SOS2 antibodies react with both human and mouse SOS2, facilitating translational research across model systems .

• Validation Data: Review available validation data including western blot images showing predicted band sizes (approximately 153-170 kDa) and application-specific controls .

• Clone Type: Consider whether polyclonal or monoclonal antibodies better suit your experimental needs. Polyclonal antibodies may offer higher sensitivity but potentially lower specificity compared to monoclonal alternatives.

How do directly HRP-conjugated antibodies compare with two-step detection systems?

While the search results primarily reference unconjugated primary antibodies used with separate HRP-conjugated secondary antibodies, understanding the comparative performance is important:

When using SOS2 antibodies with HRP-conjugated detection systems, researchers typically use dilutions of 1:6000 for the primary antibody followed by appropriate HRP-conjugated secondary antibodies for optimal signal-to-noise ratio .

How should researchers optimize western blot protocols for SOS2 detection?

Successful SOS2 detection via western blotting requires careful optimization:

• Lysate Preparation: Due to SOS2's interaction with membrane-associated proteins, use lysis buffers containing appropriate detergents to ensure complete extraction. RIPA buffer supplemented with protease inhibitors has been validated for SOS2 extraction from various cell lines including HeLa, HEK293T, and NIH3T3 .

• Protein Loading: Optimal detection typically requires 50μg of total protein per lane, though this may vary based on cell type and SOS2 expression levels .

• Antibody Concentration: For primary antibodies, concentrations of 0.1-1μg/mL have been validated, with exposure times of 30-75 seconds for chemiluminescence detection systems .

• Membrane Type: PVDF membranes have been successfully used with goat anti-human SOS2 antibodies followed by HRP-conjugated secondary antibodies .

• Positive Controls: Include lysates from cells known to express SOS2 (HeLa, HEK293T) as positive controls in experimental setups .

How can researchers effectively troubleshoot non-specific binding with SOS2 antibodies?

Non-specific binding can complicate SOS2 detection. Address this methodologically through:

• Blocking Optimization: Extend blocking time (1-2 hours) with 5% non-fat dry milk or BSA in TBST.

• Antibody Titration: Perform careful antibody titration experiments to determine the minimum concentration yielding specific signal. For western blotting, starting dilutions of 1:6000 have been effective for certain SOS2 antibodies .

• Wash Protocol Enhancement: Implement additional and longer wash steps (5-6 washes of 10 minutes each) to reduce background.

• Validation Controls: Include SOS2 knockout or knockdown samples as negative controls to distinguish specific from non-specific signals .

• Secondary Antibody Optimization: When using HRP-conjugated secondary antibodies, ensure they are matched to the host species of the primary antibody and used at appropriate dilutions.

How can SOS2 antibodies be utilized to investigate RTK-stimulated signaling pathways?

SOS2 antibodies can be powerful tools for dissecting receptor tyrosine kinase (RTK) signaling cascades:

• Co-immunoprecipitation Studies: Use SOS2 antibodies to pull down SOS2 and associated proteins from cells stimulated with growth factors. This approach can reveal dynamic interactions with adaptor proteins like Grb2 following receptor activation .

• Phosphorylation Status Analysis: Combine SOS2 immunoprecipitation with phospho-specific antibodies to assess how RTK activation affects SOS2 post-translational modifications.

• Signaling Kinetics: Employ SOS2 antibodies in time-course experiments following RTK stimulation to track the temporal dynamics of SOS2 recruitment and downstream signaling. Research has shown that SOS2 deletion significantly affects ERK signaling pathways, particularly when combined with SOS1 inhibition .

• Functional Redundancy: Use SOS2 antibodies alongside SOS1 detection to investigate compensatory mechanisms, as studies have demonstrated that either SOS1 inhibition or SOS2 deletion can significantly inhibit phosphorylated ERK in cancer cell lines .

What role does SOS2 play in cancer research and how can antibodies facilitate this investigation?

SOS2 has emerging significance in cancer biology, particularly in therapeutic resistance:

• Resistance Mechanisms: Research indicates that SOS2 deletion affects the development of resistance to targeted therapies such as osimertinib in non-small cell lung cancer (NSCLC) . SOS2 antibodies can help monitor protein expression in resistant versus sensitive cell populations.

• 3D Culture Systems: SOS2 deletion has been shown to reduce transforming growth and enhance drug-induced killing in reduced serum conditions, particularly in PC9 cells . Immunohistochemical applications of SOS2 antibodies can help visualize these effects in 3D culture models.

• Pathway Cross-talk: While SOS2 deletion significantly inhibits ERK signaling, research indicates it does not alter AKT phosphorylation . This differential effect on downstream pathways can be monitored using SOS2 antibodies in conjunction with phospho-specific antibodies against ERK and AKT.

How can SOS2 antibodies be incorporated into multiplexed detection systems?

Advanced multiplexing approaches with SOS2 antibodies enable comprehensive pathway analysis:

• Sequential Immunoblotting: Perform sequential probing of membranes with SOS2 antibodies and antibodies against other pathway components after appropriate stripping protocols.

• Multiplex Immunofluorescence: Combine SOS2 antibodies with antibodies against interacting partners or downstream effectors, using distinct fluorophores for co-localization studies.

• Bead-Based Multiplex Assays: Incorporate SOS2 antibodies into bead-based platforms for simultaneous quantification of multiple analytes in the RAS-MAPK pathway.

• Mass Cytometry: Conjugate SOS2 antibodies with metal isotopes for high-dimensional analysis of signaling networks at the single-cell level.

What considerations are important when using SOS2 antibodies in genetic manipulation studies?

When using SOS2 antibodies to assess the consequences of genetic manipulations:

• Complete Knockout Validation: Use SOS2 antibodies to confirm complete protein elimination in CRISPR-Cas9 knockout models, as demonstrated in studies examining SOS2 deletion effects on drug resistance .

• Compensatory Mechanism Detection: Monitor potential upregulation of SOS1 or other RAS-GEFs following SOS2 deletion/inhibition, as pathway adaptation is common in signaling networks .

• Isoform-Specific Detection: Ensure antibodies can distinguish between potential splice variants or isoforms to accurately assess manipulation outcomes.

• Temporal Dynamics: Consider the timing of antibody-based detection following genetic manipulation, as SOS2 has a shorter half-life compared to SOS1, which may affect experimental outcomes .