SH3GL2 Antibody

What is SH3GL2 and where is it primarily expressed?

SH3GL2 (SH3 domain containing GRB2 Like 2), also known as Endophilin A1, is a protein implicated in synaptic vesicle endocytosis and membrane curvature. It is abundantly expressed in neural tissues, as demonstrated by Western blot analyses of human brain stem, hypothalamus, and cerebellum tissues, as well as mouse and rat brain tissues . The protein contains an SH3 domain and is part of the endophilin family. It has a calculated molecular weight of 40 kDa, which is consistent with observed weights in Western blot analyses (38-40 kDa) . SH3GL2 plays critical roles in recruiting proteins to membranes with high curvature and is required for BDNF-dependent dendrite outgrowth, suggesting its importance in neuronal development and function .

What applications are SH3GL2 antibodies suitable for?

SH3GL2 antibodies are validated for multiple research applications with varying optimal dilutions:

Research has confirmed detection of SH3GL2 in human brain tissue (cerebellum), mouse brain tissue, and rat brain tissue using Western blot . When conducting Western blot experiments, researchers have successfully used protocols with PVDF membranes probed with 1 μg/mL of antibody, followed by HRP-conjugated secondary antibodies, under reducing conditions . For optimal results, researchers should determine specific dilutions based on their experimental conditions and sample types .

How should SH3GL2 antibodies be stored and handled for maximum efficacy?

For optimal longevity and performance of SH3GL2 antibodies, researchers should aliquot the antibody upon receipt and store at -20°C, avoiding repeated freeze/thaw cycles that can degrade antibody performance . Most commercial antibodies are provided in a buffer containing PBS (pH 7.3), sodium azide (0.02%) as a preservative, and glycerol (50%) to prevent freezing damage . When working with the antibody, allow it to equilibrate to room temperature before opening to prevent condensation that could introduce contaminants. For long-term storage projects, monitoring antibody performance through positive controls is recommended to assess potential deterioration over time.

How can SH3GL2 antibodies be utilized to study its role in cancer biology?

SH3GL2 has been identified as frequently deleted in non-small cell lung cancer (NSCLC), suggesting a potential tumor suppressor role . Researchers investigating SH3GL2 in cancer can employ multiple methodological approaches:

• Loss of Heterozygosity (LOH) Analysis: Studies have documented LOH of SH3GL2 in 51% (49/97) of informative NSCLC cases, indicating genetic alterations at the SH3GL2 locus as a potential mechanism of inactivation .

• Protein Expression Analysis: Western blot and immunohistochemistry can be used to assess SH3GL2 protein levels. Research has shown significantly reduced SH3GL2 protein expression in 71% (43/60) of primary NSCLC tumors .

• Functional Studies: Researchers have demonstrated that forced expression of wild-type SH3GL2 in NSCLC cell lines results in:

  • Increased EGFR internalization and degradation

  • Decreased cellular growth in vitro (p=0.0015-0.030) and in vivo (p=0.016)

  • Reduced invasion capabilities (p=0.029-0.049)

  • Decreased colony formation (p=0.023-0.039)

These methodological approaches allow researchers to comprehensively evaluate SH3GL2's role in cancer progression and its potential as a therapeutic target.

What control experiments should be included when using SH3GL2 antibodies in EGFR signaling studies?

When investigating SH3GL2's effects on EGFR signaling, comprehensive controls are essential for data validation:

• Positive Controls: Include tissue/cell lysates known to express SH3GL2 (e.g., brain tissues) to confirm antibody functionality .

• Negative Controls: Use SH3GL2-null or knockdown cells along with isotype-matched control antibodies to confirm specificity.

• EGF Stimulation Time Course: Since SH3GL2 affects EGFR internalization, researchers should include a time course of EGF stimulation (e.g., 0, 5, 15, 30 minutes, 6 hours) to capture dynamic changes. Research has shown marked differences in EGFR internalization between SH3GL2-transfected and control cells following EGF treatment .

• Downstream Effector Verification: Monitor changes in phosphorylation of downstream EGFR targets (AKT, STAT3, PI3K) as these have been shown to decrease with SH3GL2 overexpression .

• Co-immunoprecipitation Controls: When studying SH3GL2-protein interactions, include both input control and IgG control precipitations.

• Cross-reactivity Assessment: Test for potential cross-reactivity with related proteins (SH3GL1, SH3GL3) when using antibodies that might recognize multiple family members .

What are the methodological considerations for investigating SH3GL2 in endocytic trafficking?

SH3GL2's role in endocytosis requires specific methodological approaches:

• Immunofluorescence Colocalization Studies: Use dual labeling with endocytic markers (e.g., EEA1 for early endosomes, LAMP1 for lysosomes) and SH3GL2 antibody. Optimal dilutions for immunofluorescence range from 1/50 to 1/200 .

• Live Cell Imaging: For tracking dynamics of SH3GL2-mediated endocytosis, consider using fluorescently tagged SH3GL2 constructs alongside endocytic cargo markers.

• Receptor Internalization Assays: As demonstrated in research, EGF-stimulated EGFR internalization can be visualized and quantified in SH3GL2-transfected versus control cells. Researchers have successfully used this approach to show increased EGFR internalization in SH3GL2-expressing cells .

• Subcellular Fractionation: To biochemically assess SH3GL2 localization during endocytosis, membrane and cytosolic fractions can be separated and analyzed by Western blot.

• Blocking Specific Endocytic Pathways: Use inhibitors of different endocytic pathways (e.g., dynamin inhibitors) to determine which pathways involve SH3GL2.

How can researchers troubleshoot non-specific binding when using SH3GL2 antibodies?

When encountering non-specific binding with SH3GL2 antibodies, implement these methodological approaches:

• Optimize Blocking Conditions: Research protocols indicate 5% non-fat dry milk in TBST as an effective blocking buffer for Western blot applications . Consider testing alternative blocking agents (BSA, casein) if background persists.

• Titrate Antibody Concentration: Start with the manufacturer's recommended dilution (1/500-1/2000 for WB) and adjust as needed based on signal-to-noise ratio .

• Increase Washing Steps: Extend washing durations or add additional washes with TBST to reduce background.

• Use Validated Positive Controls: Include lysates from tissues known to express SH3GL2 (brain tissues) as positive controls .

• Pre-adsorption Control: If available, pre-incubate the antibody with recombinant SH3GL2 protein before application to confirm specificity of binding.

• Check Cross-Reactivity: Some antibodies may detect multiple endophilin family members (SH3GL1, SH3GL2, SH3GL3); verify whether your antibody is specific to SH3GL2 or detects multiple family members .

What are the key considerations for optimizing SH3GL2 detection in different tissue types?

Tissue-specific optimization is crucial for successful SH3GL2 detection:

• Brain Tissue Processing: Since SH3GL2 is highly expressed in neural tissues, special attention to tissue preservation is needed. Flash-freezing is preferable to formalin fixation for maintaining protein integrity.

• Epitope Accessibility: For immunohistochemistry or immunofluorescence in fixed tissues, consider antigen retrieval methods. Heat-induced epitope retrieval may be necessary, especially for formalin-fixed tissues.

• Species-Specific Validation: Confirm antibody reactivity with your species of interest. Available data confirms reactivity with human, mouse, and rat samples, but cross-reactivity with other species should be determined experimentally .

• Loading Control Selection: For Western blot quantification, select appropriate loading controls based on tissue type. For brain tissue, neuronal markers like β-III-tubulin may be more appropriate than general housekeeping proteins.

• Extraction Buffer Composition: For tissues with high lipid content like brain, include detergents that efficiently solubilize membrane proteins without denaturing the epitope recognized by the antibody.

• Detection System Sensitivity: For tissues with lower SH3GL2 expression, consider using more sensitive detection systems such as chemiluminescent substrates with enhanced sensitivity or fluorescent secondary antibodies.

How can SH3GL2 antibodies be used to investigate synaptic vesicle endocytosis?

SH3GL2 plays a critical role in synaptic vesicle endocytosis, and researchers can investigate this function using several approaches:

• Synaptosomes Preparation and Analysis: Isolate synaptosomes from brain tissue and analyze SH3GL2 localization and interaction partners during stimulated endocytosis. Western blot detection can be performed using antibody dilutions of 1/500 to 1/2000 .

• Primary Neuronal Cultures: In cultured neurons, use immunofluorescence (recommended dilution 1/50 - 1/200) to visualize SH3GL2 localization at synapses, particularly following stimulation protocols that trigger synaptic vesicle recycling.

• Co-localization with Synaptic Markers: Perform double immunostaining with SH3GL2 antibodies and markers for synaptic vesicles (synaptophysin) or active zones (bassoon).

• BDNF-Dependent Processes: Since SH3GL2 is required for BDNF-dependent dendrite outgrowth , researchers can use SH3GL2 antibodies to investigate the role of this protein in BDNF-NTRK2 early endocytic trafficking and signaling from early endosomes.

• Ultrastructural Localization: Immuno-electron microscopy can provide detailed information about SH3GL2 localization at the ultrastructural level, particularly around synaptic vesicles and membrane invaginations.

What methodological approaches can be used to study SH3GL2 in membrane curvature induction?

SH3GL2's role in inducing membrane curvature can be studied through these methodological approaches:

• Recombinant Protein Studies: Purify recombinant SH3GL2 protein to study its direct effect on artificial membrane systems. The BAR domain of SH3GL2 can be specifically targeted in these studies.

• Liposome Tubulation Assays: Incubate purified SH3GL2 with fluorescently labeled liposomes and observe membrane tubulation using fluorescence microscopy.

• Live Cell Imaging with Membrane Markers: In cells expressing fluorescently tagged SH3GL2, monitor membrane deformation during endocytic events using high-resolution microscopy techniques.

• Structure-Function Analysis: Generate SH3GL2 mutants with alterations in the BAR domain and assess their impact on membrane curvature using antibodies against SH3GL2 to detect proper expression and localization.

• Atomic Force Microscopy: Use AFM to directly visualize SH3GL2-induced membrane deformations on supported lipid bilayers.

• Cryo-Electron Microscopy: Visualize SH3GL2-membrane interactions at near-atomic resolution to understand the structural basis of membrane curvature induction.

How might SH3GL2 antibodies be utilized in cancer biomarker research?

Given SH3GL2's frequent deletion in NSCLC and its potential tumor suppressor role , researchers can explore its utility as a cancer biomarker:

• Tissue Microarray Analysis: Develop immunohistochemistry protocols using SH3GL2 antibodies to screen large cohorts of tumor samples for expression patterns. Research has shown significantly low SH3GL2 protein expression in 71% of primary NSCLC tumors .

• Correlation with Clinical Outcomes: Analyze SH3GL2 expression levels in relation to patient survival, tumor stage, and treatment response.

• Multi-marker Panels: Combine SH3GL2 with other EGFR pathway markers to create predictive/prognostic panels, as research has shown SH3GL2 regulates EGFR signaling through increased receptor internalization and degradation .

• Liquid Biopsy Development: Explore whether SH3GL2 or its fragments can be detected in circulation and serve as non-invasive biomarkers.

• Genetic-Protein Expression Correlation: Compare SH3GL2 genetic alterations (LOH, which occurs in 51% of NSCLC cases) with protein expression to determine the relationship between genetic events and protein levels .

• Therapeutic Response Prediction: Investigate whether SH3GL2 expression levels predict response to EGFR-targeted therapies, given its role in modulating EGFR signaling.

What considerations are important when studying SH3GL2 interactions with other proteins?

When investigating SH3GL2 protein interactions, researchers should consider:

• Co-immunoprecipitation Protocols: Optimize lysis conditions that preserve protein-protein interactions. For SH3GL2, which interacts with membrane components, include appropriate detergents that solubilize membranes without disrupting interactions.

• Reciprocal IP Verification: Perform IP with antibodies against both SH3GL2 and its suspected interaction partners (such as USP9X and β-catenin, which have been shown to be downregulated in SH3GL2-transfected cells) .

• Domain-Specific Interactions: The SH3 domain of SH3GL2 mediates many protein interactions. Use domain deletion mutants to map interaction domains specifically.

• Cross-linking Approaches: For transient interactions, consider chemical cross-linking before immunoprecipitation.

• Proximity Ligation Assays: This technique can visualize protein-protein interactions in situ at endogenous expression levels, providing spatial information about where interactions occur.

• EGFR Pathway Interactions: When studying SH3GL2's effects on EGFR signaling, monitor interactions with components of the endocytic machinery and downstream effectors including AKT, STAT3, and PI3K, which show reduced activation in SH3GL2-expressing cells .