SHC2 Antibody

What is SHC2 and what functional domains characterize this protein?

SHC2 (also known as SHC-transforming protein 2, Protein Sck, SHC-transforming protein B, or SH2 domain protein C2) is a 62kDa cytoplasmic signaling adaptor protein that contains several critical functional domains . The protein structure includes an N-terminal phosphotyrosine binding (PTB) domain, a central collagen homology (CH) domain, and a C-terminal Src homology 2 (SH2) domain . These domains work in concert to facilitate SHC2's primary function - coupling activated growth factor receptors to downstream signaling pathways in neurons . The SH2 domain is particularly important as it enables SHC2 to bind specifically to tyrosine-phosphorylated proteins that participate in signal transduction cascades . Gene Ontology annotations associate SHC2 with numerous signaling pathways including Ras protein signal transduction, insulin receptor signaling, and nerve growth factor receptor signaling .

In which tissues and cell types is SHC2 predominantly expressed?

SHC2 exhibits a tissue-specific expression pattern with predominant expression in neural tissues, consistent with its role in neuronal signaling pathways . Human SHC2 transcripts are present at high levels in liver, pancreas, prostate, and ovary . The protein has been detected in multiple human tissues through immunohistochemical analysis, including pancreas, cerebral cortex, prostate, and lymph node tissues . At the cellular level, SHC2 expression has been confirmed in several cell lines including RT4 (human urinary bladder cancer), U-251 MG (human brain glioma), and vascular endothelial cells . In vascular endothelial cells specifically, SHC2 participates in VEGF-induced signal transduction, where it is recruited to tyrosine-1175 of the kinase insert domain-containing receptor (KDR) following VEGF stimulation . This tissue and cell-type distribution reflects SHC2's specialized roles in neuronal and endothelial signaling contexts.

What types of SHC2 antibodies are commercially available for research?

Several types of SHC2 antibodies are available for research applications, each with distinct characteristics suited to different experimental needs :

These antibodies differ in their target epitopes, which can impact their effectiveness in different applications and experimental conditions . The mouse monoclonal antibody (clone rEF01/6C7) demonstrates cross-reactivity with rat and mouse SHC2 in addition to human, though antibody reactivity may vary between species . Researchers should select an antibody based on their specific application requirements, target species, and the region of SHC2 they wish to detect.

How should I select the appropriate SHC2 antibody for my specific research application?

Selecting the optimal SHC2 antibody requires consideration of multiple factors to ensure experimental success . First, verify application compatibility - different antibodies are validated for specific techniques such as Western blotting or immunohistochemistry . For instance, the Mouse anti-human SHC2 (clone rEF01/6C7) is validated for Western blotting at 1/1,000 dilution, while the Rabbit Polyclonal SHC2 antibody (ab243385) is validated for both Western blotting and IHC-P . Second, confirm species reactivity matches your experimental model . The mouse monoclonal antibody (clone rEF01/6C7) shows cross-reactivity with rat and mouse SHC2 in addition to human targets .

Consider the specific epitope recognition pattern of each antibody option . Some antibodies target the CH1 and SH2 domains, while others recognize the C-terminal region (aa 400-550) . This becomes particularly important when studying specific domains or if certain regions might be masked due to protein interactions or conformational changes. Weigh the advantages of monoclonal versus polyclonal antibodies based on your experimental needs . Monoclonal antibodies offer high specificity for a single epitope, while polyclonal antibodies provide stronger signals by recognizing multiple epitopes, potentially increasing detection sensitivity . Finally, prioritize antibodies with robust validation data in your application of interest, including published results demonstrating specific detection of SHC2 in relevant experimental systems .

What controls should I use when working with SHC2 antibodies?

Implementing appropriate controls is essential for ensuring reliable results when working with SHC2 antibodies . For positive controls, use tissues or cell lines with confirmed SHC2 expression . Based on published data, suitable positive controls include human brain tissue (particularly cerebral cortex), RT4 cell lysate, and U-251 MG cell lysate . For negative controls, include a primary antibody omission control (all reagents except primary antibody) and an isotype control matching the primary antibody's isotype (e.g., IgG2a for mouse monoclonal clone rEF01/6C7) .

For technical validation, include a blocking peptide competition assay where the SHC2 antibody is pre-incubated with the immunizing peptide before application to the sample . This should substantially reduce or eliminate specific staining. In Western blotting applications, always include appropriate loading controls (e.g., β-actin, GAPDH) to verify equal protein loading across samples . For advanced validation, consider using siRNA knockdown of SHC2 to confirm antibody specificity - signal reduction proportional to knockdown efficiency strongly supports antibody specificity . When evaluating phosphorylation-dependent events, include both stimulated and unstimulated samples, as SHC2 phosphorylation increases significantly following growth factor stimulation (e.g., VEGF treatment in endothelial cells) . These controls collectively enhance confidence in the specificity and reliability of SHC2 antibody-based experimental results.

What are the optimal conditions for using SHC2 antibodies in Western blotting?

Achieving optimal results with SHC2 antibodies in Western blotting requires careful attention to protocol details . For sample preparation, use standard cell lysis buffers containing protease inhibitors to prevent degradation, adding phosphatase inhibitors if studying SHC2 phosphorylation . Denature samples at 95°C for 5 minutes in SDS-PAGE loading buffer before loading. Use 10-12% SDS-PAGE gels for optimal separation, as SHC2 has an expected molecular weight of approximately 62 kDa .

For antibody incubation, block membranes with 5% non-fat dry milk or 5% BSA in TBST for 1 hour at room temperature . When using the Mouse anti-human SHC2 (clone rEF01/6C7), apply at a 1/1,000 dilution as validated in published protocols . For the Rabbit Polyclonal SHC2 antibody (ab243385), use at 0.4 μg/mL concentration as demonstrated in published Western blot data . Incubate with primary antibody overnight at 4°C or for 2 hours at room temperature, followed by thorough washing (3-5 times with TBST) . Use an appropriate HRP-conjugated secondary antibody matched to the primary antibody's host species and isotype .

Published Western blot data shows successful detection of SHC2 in various human samples including RT4 cell lysate, U-251 MG cell lysate, human plasma (IgG/HSA depleted), human liver tissue lysate, and human tonsil tissue lysate . The expected band size is 62 kDa, though post-translational modifications may result in slight variations in migration patterns . Occasional additional bands may represent phosphorylated forms or proteolytic fragments .

How can I optimize immunohistochemistry protocols for SHC2 detection?

For optimal immunohistochemical detection of SHC2, several key protocol steps require careful optimization . Begin with proper tissue preparation: fix tissues in 10% neutral buffered formalin, embed in paraffin, and section at 4-6 μm thickness . Antigen retrieval is critical for SHC2 detection in formalin-fixed tissues; heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is recommended . Perform this step using a microwave or pressure cooker for 15-20 minutes to ensure complete epitope exposure.

For blocking and antibody incubation, first block endogenous peroxidase with 3% H₂O₂ for 10 minutes, then block non-specific binding with 5-10% normal serum from the secondary antibody species . When using the Rabbit Polyclonal SHC2 antibody (ab243385), a 1/50 dilution has been validated for IHC-P applications . Incubate with primary antibody overnight at 4°C or for 1-2 hours at room temperature for optimal signal generation . After thorough washing, apply an appropriate secondary antibody system - either standard two-step detection or more sensitive polymer-based detection systems for enhanced signal .

Published IHC data demonstrates successful SHC2 detection in multiple human tissues including pancreas, cerebral cortex, prostate, and lymph node . Each tissue may require slight protocol modifications for optimal results. Always include positive control tissues with known SHC2 expression (such as cerebral cortex) and negative controls (primary antibody omission) in each staining run to verify staining specificity .

How can I investigate SHC2's role in neurotrophin signaling pathways?

Investigating SHC2's function in neurotrophin signaling requires multifaceted experimental approaches . Begin with expression analysis to characterize SHC2 expression patterns in different neuronal populations using the validated SHC2 antibodies . Immunohistochemistry with the Rabbit Polyclonal SHC2 antibody (ab243385) has been successful in detecting SHC2 in human cerebral cortex tissue, providing spatial information about expression patterns . For functional studies, implement gene knockdown using siRNA or CRISPR-Cas9 to reduce SHC2 expression, then assess the effects on neurotrophin-dependent processes including neurite outgrowth, cell survival, and neuronal differentiation .

To analyze signaling dynamics, stimulate neurons with neurotrophins (NGF, BDNF, NT-3, NT-4/5) and monitor SHC2 phosphorylation using Western blotting . The Mouse anti-human SHC2 antibody (clone rEF01/6C7) at 1/1,000 dilution can detect total SHC2 levels in these experiments . Assess activation of downstream pathways including Ras/MAPK, PI3K/Akt, and PLCγ pathways following stimulation in both control and SHC2-depleted conditions . For protein interaction studies, perform co-immunoprecipitation experiments to investigate SHC2's association with Trk receptors and downstream effectors such as GRB2 and SOS . This approach reveals how SHC2 functions as an adaptor protein in assembling signaling complexes following neurotrophin stimulation.

The combined results from these approaches can address key research questions including how SHC2 contributes to neurotrophin specificity, the interplay between SHC2 and other SHC family members in neuronal signaling, and how SHC2 regulates the balance between different downstream signaling pathways .

What approaches can be used to study SHC2 phosphorylation dynamics?

Studying SHC2 phosphorylation dynamics requires specialized techniques that capture this critical post-translational modification . Since SHC2 functions as a signaling adaptor protein that couples activated growth factor receptors to downstream effectors, its phosphorylation state is a key regulatory mechanism . For baseline phosphorylation analysis, implement Western blotting with phospho-specific antibodies that recognize specific phosphorylated tyrosine residues on SHC2 . When performing these experiments, include phosphatase inhibitors in lysis buffers to preserve phosphorylation status, and compare samples under basal conditions versus after stimulation with appropriate ligands (e.g., neurotrophins for neuronal cells, VEGF for endothelial cells) .

For temporal dynamics, perform time-course experiments after stimulation, collecting samples at multiple timepoints (30 seconds, 2 minutes, 5 minutes, 15 minutes, 30 minutes, 1 hour) to track the phosphorylation and dephosphorylation kinetics . In vascular endothelial cells, SHC2 becomes phosphorylated following VEGF stimulation, with recruitment to tyrosine-1175 of the KDR receptor . This system provides an excellent model for studying phosphorylation dynamics in a physiologically relevant context. To identify the specific phosphorylation sites, immunoprecipitate SHC2 after cell stimulation using validated antibodies and analyze the precipitated protein by mass spectrometry .

For functional validation of specific phosphorylation sites, create phosphorylation site mutants (Y to F mutations) and assess their impact on downstream signaling and cellular functions . This approach can reveal which phosphorylation events are necessary for specific SHC2-mediated functions. Collectively, these approaches provide comprehensive insights into how SHC2 phosphorylation regulates its adaptor function in growth factor signaling pathways.

How can I differentiate between SHC family members in my experiments?

Differentiating between SHC family members requires careful experimental design due to their structural similarities . The SHC family includes SHC1 (ubiquitously expressed), SHC2 (predominantly in neuronal tissues), SHC3 (brain-specific), and SHC4 (expressed in melanocytes and some cancer cells) . For antibody-based differentiation, select antibodies that specifically recognize unique epitopes in each SHC family member . The Mouse anti-human SHC2 antibody (clone rEF01/6C7) recognizes SHC2 specifically, but validation against other SHC family members is recommended to confirm absolute specificity .

For expression analysis, perform qRT-PCR with primers specific to each SHC family member to distinguish their expression patterns across tissues and cell types . This approach can be particularly useful when antibody cross-reactivity is a concern. Consider tissue-specific expression patterns when designing experiments - SHC2 shows predominant expression in neural tissues, liver, pancreas, prostate, and ovary, which can help distinguish it from other family members .

Why might I observe multiple bands when detecting SHC2 by Western blot?

Multiple bands in SHC2 Western blots can arise from several biological and technical sources . The primary expected band for SHC2 is approximately 62 kDa, but additional bands may appear due to post-translational modifications, particularly phosphorylation . In stimulated samples (e.g., after growth factor treatment), phosphorylated forms of SHC2 may appear as slower-migrating bands with slightly higher apparent molecular weight . To confirm phosphorylation-induced shifts, treat a portion of your lysate with phosphatase before electrophoresis - if the higher molecular weight bands disappear, this indicates they represent phosphorylated forms .

Alternative splicing could generate SHC2 isoforms of different sizes . Though the primary literature on SHC2 doesn't extensively document multiple splice variants (unlike SHC1 which has three well-characterized isoforms), this possibility should be considered . Proteolytic processing during sample preparation can generate fragment bands. To minimize this, add complete protease inhibitor cocktails to lysis buffers and process samples quickly at 4°C .

Cross-reactivity with other SHC family members can also generate unexpected bands . The SHC family includes several members with similar molecular weights: SHC1 (46, 52, 66 kDa), SHC2 (62 kDa), SHC3 (64 kDa), and SHC4 (68 kDa) . To distinguish between these possibilities, validate your antibody using recombinant SHC proteins or lysates from cells with confirmed expression of specific SHC family members . Include SHC2 knockdown controls to confirm which bands specifically represent SHC2 .

What are the best practices for storing and handling SHC2 antibodies?

Proper storage and handling of SHC2 antibodies is critical for maintaining their performance and extending their usable lifespan . Most SHC2 antibodies, including the Mouse anti-human SHC2 antibody (clone rEF01/6C7), are supplied as purified IgG in liquid format . These should be stored according to manufacturer recommendations, typically at -20°C for long-term storage . The antibody buffer solution usually contains phosphate buffered saline with preservatives such as 0.09% sodium azide to maintain stability .

Avoid repeated freezing and thawing as this may denature the antibody and reduce its performance . Storage in frost-free freezers is not recommended due to temperature fluctuations that can damage antibody structure . When working with the antibody, aliquot the stock into smaller volumes upon first thaw to minimize freeze-thaw cycles. For short-term storage (1-2 weeks), antibodies can typically be kept at 4°C, but refer to specific product documentation for confirmation .

When diluting antibodies for experimental use, prepare fresh working dilutions on the day of the experiment whenever possible . Use high-quality, clean buffers for dilution to prevent contamination. Most SHC2 antibodies come with a guarantee period (typically 12 months from the date of dispatch) when stored as recommended . Before using antibodies that have been stored for extended periods, it's advisable to test their performance in a validation experiment comparing them to a fresh or previously functional batch . Following these handling practices will help ensure consistent results and maximize the useful life of SHC2 antibodies.

How can I validate the specificity of my SHC2 antibody?

Comprehensive validation of SHC2 antibody specificity requires multiple complementary approaches . Begin with genetic approaches by testing the antibody in SHC2 knockdown or knockout models . A significant reduction in signal intensity proportional to the degree of knockdown provides strong evidence of specificity . For molecular validation, perform Western blotting with recombinant SHC2 protein to confirm detection at the expected molecular weight (approximately 62 kDa) . Peptide competition assays, where the antibody is pre-incubated with the immunizing peptide before application to samples, should substantially reduce or eliminate specific signals .

For orthogonal validation, compare protein detection results with mRNA expression data from qRT-PCR or RNA-seq analysis . Correlation between protein and mRNA levels across different tissues or experimental conditions supports antibody specificity. Compare results from multiple antibodies targeting different SHC2 epitopes - concordant detection patterns strongly support specificity . For the Mouse anti-human SHC2 antibody (clone rEF01/6C7) which recognizes domains CH1 and SH2, compare with antibodies recognizing other regions of SHC2 .

Application-specific validation is also important . For Western blotting, verify the correct molecular weight (approximately 62 kDa) and confirm band disappearance after knockdown . For immunohistochemistry, compare staining patterns with known SHC2 expression profiles - the antibody should show strong staining in tissues known to express SHC2 (cerebral cortex, pancreas, prostate, liver) . Cross-reactivity testing against other SHC family members can be performed using recombinant proteins or cell lines with defined expression patterns . This comprehensive validation approach ensures confidence in antibody specificity across different experimental contexts.

How can SHC2 antibodies be utilized in cancer research?

SHC2 antibodies offer valuable tools for investigating cancer-related signaling pathways, particularly in neurological and vascular malignancies . Immunohistochemical analysis using SHC2 antibodies can assess expression patterns across different tumor types and correlate expression with clinical parameters and patient outcomes . Published data demonstrates successful SHC2 detection in human cancer cell lines including RT4 (urinary bladder cancer) and U-251 MG (brain glioma) . Researchers can employ a standardized IHC protocol using Rabbit Polyclonal SHC2 antibody (ab243385) at 1/50 dilution for formalin-fixed, paraffin-embedded tumor samples .

For mechanistic studies, SHC2 antibodies can investigate how this adaptor protein contributes to oncogenic signaling cascades . In vascular tumors, SHC2's role in VEGF-induced signal transduction is particularly relevant, as it participates in angiogenic pathways critical for tumor growth and metastasis . Using co-immunoprecipitation approaches with SHC2 antibodies, researchers can identify novel interaction partners in cancer cells that may represent therapeutic targets . Western blotting applications can assess how SHC2 phosphorylation status changes in response to targeted therapies, potentially identifying biomarkers of treatment response .

Analysis of SHC2's participation in receptor tyrosine kinase signaling pathways that are frequently dysregulated in cancer (including MAPK signaling cascades) can reveal new therapeutic vulnerabilities . The relationship between SHC2 and other SHC family members in cancer contexts is particularly interesting, as SHC1 has established roles in oncogenic signaling while SHC4 has been implicated specifically in melanoma progression . SHC2 antibodies enable comparative analysis of these family members across cancer types and stages.

What role does SHC2 play in receptor tyrosine kinase signaling pathways?

SHC2 functions as a critical adaptor protein in receptor tyrosine kinase (RTK) signaling pathways, coupling activated receptors to downstream effectors . As a cytoplasmic signaling adaptor protein containing a Src homology 2 (SH2) domain, SHC2 can bind directly to phosphorylated tyrosine residues on activated RTKs . This interaction is well-documented in neurotrophin-activated Trk receptors in cortical neurons, where SHC2 participates in signal transduction pathways critical for neuronal development and function . In vascular endothelial cells, SHC2 is recruited to tyrosine-1175 of the kinase insert domain-containing receptor (KDR) following VEGF stimulation, highlighting its role in angiogenic signaling .

Following RTK binding, SHC2 becomes tyrosine phosphorylated, creating binding sites for other signaling molecules . SHC2 interacts with proteins such as GRB2 and SOS, fostering signal transduction that activates downstream pathways including the Ras-MAPK cascade . Gene ontology annotations confirm SHC2's participation in multiple RTK-mediated pathways, including Ras protein signal transduction, insulin receptor signaling, and nerve growth factor receptor signaling .

SHC2's domain architecture is perfectly suited for its adaptor function in RTK signaling . The N-terminal PTB domain can bind phosphotyrosine residues in a different context than the SH2 domain, allowing potentially simultaneous interactions with multiple signaling proteins . The central collagen homology (CH) domain may facilitate interactions with additional partners, while the C-terminal SH2 domain recognizes specific phosphotyrosine-containing motifs on activated receptors . This multi-domain structure enables SHC2 to function as a versatile scaffold that organizes signaling complexes downstream of activated RTKs .