sh3bgrl2 Antibody

What is SH3BGRL2 and why is it significant for cancer research?

SH3BGRL2 is a member of the SH3BGR protein family that contains a highly conserved proline-rich domain involved in interactions with proteins containing specific binding modules such as SH3, WW, and EVH1 domains . Recent studies have revealed its dual role in cancer biology, particularly in breast cancer where it suppresses tumor growth but enhances metastatic capacity . In clear cell renal cell carcinoma (ccRCC), SH3BGRL2 functions as a tumor suppressor through the Hippo/TEAD1-Twist1 signaling pathway . The protein's observed molecular weight ranges from 12-17 kDa when detected by Western blot .

What applications are most effective for SH3BGRL2 antibody detection?

Based on validated research protocols, SH3BGRL2 antibodies have demonstrated efficacy in multiple applications:

For optimal results, researchers should validate antibody performance in their specific experimental system, as reactivity may vary between human and mouse samples .

How should I select the most appropriate SH3BGRL2 antibody for my experiments?

Selection should be guided by:

• Target species relevance: Confirm reactivity with your experimental model (human, mouse, rat)

• Epitope location: Antibodies targeting different regions (e.g., AA 41-107) may perform differently depending on protein conformation and interactions

• Application compatibility: Verify validation data for your intended application (WB, IHC, ELISA, IF)

• Conjugation requirements: Select between unconjugated antibodies or those with specific conjugates (biotin, fluorophores) based on your detection system

• Clonality consideration: Polyclonal antibodies offer broader epitope recognition, while monoclonal antibodies provide higher specificity

What are the recommended storage and handling protocols for SH3BGRL2 antibodies?

For maximum stability and performance:

• Store at -20°C in the manufacturer's recommended buffer (typically PBS with 0.02% sodium azide and 50% glycerol, pH 7.3)

• Avoid repeated freeze/thaw cycles by preparing single-use aliquots

• DO NOT ALIQUOT certain formulations (follow manufacturer specifications)

• Monitor antibody performance regularly as activity may decrease over time, with typical validity periods of 12 months

• Prior to use, allow antibody to equilibrate to room temperature and mix gently to ensure homogeneity

How can I investigate SH3BGRL2's dual function in cancer progression and metastasis?

SH3BGRL2 exhibits context-dependent functions in cancer biology that require sophisticated experimental approaches:

Recommended methodology for functional analysis:

• Expression modulation:

  • Use lentiviral vectors (e.g., pCDH-CMV-MCSEF1-Puro) for stable overexpression

  • Apply shRNA in GIPZ lentiviral vectors for knockdown studies

  • Validate expression changes via qPCR and Western blot using appropriate antibodies

• Functional assays:

  • Proliferation: Cell counting, colony formation assays

  • Metastasis: Migration/invasion assays, in vivo lung metastasis models

• Mechanistic dissection:

  • Examine interaction with spectrin cytoskeletal proteins (SPTAN1, SPTBN1) through co-immunoprecipitation

  • Analyze TGF-β1 pathway activation using inhibitors (SB431542, SIS3)

  • Investigate Hippo/TEAD1-Twist1 signaling in different cancer contexts

When interpreting results, consider that SH3BGRL2 suppresses breast cancer cell proliferation but enhances metastatic capacity, suggesting tissue-specific regulatory mechanisms .

What strategies enable reliable detection of SH3BGRL2 in patient samples for prognostic studies?

Clinical investigations of SH3BGRL2 require rigorous methodological approaches:

• Multi-level expression analysis:

  • mRNA: RT-PCR or RNA-sequencing with appropriate normalization

  • Protein: Western blot with validated antibodies at 1:500-1:5000 dilution

  • Tissue localization: IHC on paraffin-embedded sections (1:20-1:200 dilution)

• Patient cohort considerations:

  • Include matched tumor-normal pairs when possible

  • Stratify by cancer subtype (e.g., luminal, HER2+, TNBC for breast cancer)

  • Correlate with clinical parameters (stage, grade, metastasis status)

• Analytical approaches:

  • Immunohistochemical scoring systems for prognostic significance

  • Kaplan-Meier survival analysis with appropriate statistical validation

  • Integration with existing databases (TCGA, CPTAC) for validation

Studies have shown that SH3BGRL2 downregulation occurs in 62.5-92.9% of breast tumors and correlates with reduced survival in ccRCC (hazard ratio = 0.329) .

What are the methodological challenges in studying SH3BGRL2 protein interactions and how can they be addressed?

Investigating SH3BGRL2's protein-protein interactions presents several technical challenges:

• Optimal immunoprecipitation protocols:

  • Use anti-Flag magnetic beads for tagged constructs in overexpression systems

  • Apply stringent washing conditions to reduce non-specific binding

  • Include appropriate negative controls (e.g., empty vector expression)

• Mass spectrometry identification:

  • Resolve immunoprecipitated proteins by SDS-PAGE with Coomassie Blue staining

  • Perform LC-MS/MS analysis with high confidence parameters (≥95%)

  • Validate interactions using SEQUEST and Trans Proteomic Pipeline software

• Functional validation approaches:

  • Chromatin immunoprecipitation to detect transcriptional regulation effects

  • Luciferase reporter assays for promoter activity analysis

  • siRNA-mediated knockdown of interaction partners (e.g., SPTAN1, SPTBN1) for functional rescue experiments

• Subcellular localization studies:

  • Immunofluorescence microscopy to detect co-localization

  • Cell fractionation followed by immunoblotting to determine compartmentalization

  • Live-cell imaging with fluorescently tagged constructs

How do expression patterns of SH3BGRL2 differ across cancer types and what are the implications for antibody selection?

SH3BGRL2 exhibits distinct expression patterns across different cancers with important methodological considerations:

• Expression profile comparison:

  • Breast cancer: Downregulated in 92.9% of tumors at mRNA level and 67.9% at protein level

  • ccRCC: Reduced expression in 62.5% of cases with progressive decrease in higher grades

  • Varies across molecular subtypes, requiring subtype-specific analysis

• Tissue-specific antibody validation:

  • Validate antibody performance in specific cancer tissue context

  • Optimize antibody concentration for each tissue type (1:20-1:200 for IHC)

  • Include appropriate positive controls (e.g., placenta, liver for human samples)

• Analytical considerations:

  • Correct for tissue-specific background staining in immunohistochemistry

  • Account for potential post-translational modifications affecting antibody recognition

  • Consider protease inhibitors during sample preparation to prevent degradation

• Comparative methodology:

  • RNA-sequencing and protein expression should be analyzed in parallel to identify discordant regulation

  • Compare with public databases (TCGA, CPTAC) for validation across cancer types

  • Consider laser-capture microdissection for heterogeneous samples to improve specificity

What controls are essential when using SH3BGRL2 antibodies in experimental protocols?

Rigorous experimental design requires comprehensive controls:

• Antibody validation controls:

  • Positive tissue controls: Human placenta, human liver, human brain tissue

  • Negative controls: Antibody diluent only, isotype control (rabbit IgG)

  • Peptide competition assay to confirm specificity

  • siRNA/shRNA knockdown samples to verify signal reduction

• Western blot-specific controls:

  • Loading control (β-actin, GAPDH) for normalization

  • Molecular weight marker to confirm the 12-17 kDa band

  • Recombinant SH3BGRL2 protein as positive control

  • Concentration gradient to establish linear detection range

• IHC/IF controls:

  • Serial dilution series to determine optimal antibody concentration

  • Comparison of different fixation methods (paraffin, frozen sections)

  • Secondary antibody-only controls to assess background

  • Known positive tissue sections at multiple magnifications (10x, 40x)

How can I troubleshoot inconsistent results when working with SH3BGRL2 antibodies?

When encountering variable or unexpected results:

• Western blot troubleshooting:

  • Protein degradation: Add protease inhibitors during sample preparation

  • Multiple bands: Validate with recombinant protein control, consider post-translational modifications

  • Weak signal: Increase protein loading, decrease antibody dilution, extend exposure time

  • High background: Increase blocking time/concentration, use more stringent washing

• IHC optimization strategies:

  • Signal variability: Standardize fixation time and conditions

  • Background staining: Optimize blocking conditions, reduce antibody concentration

  • False negatives: Test antigen retrieval methods (heat-induced vs. enzymatic)

  • Tissue-specific issues: Adjust protocol for different tissue types (e.g., liver vs. placenta)

• Experimental design adjustments:

  • Use multiple antibodies targeting different epitopes to confirm results

  • Complement antibody detection with mRNA analysis (qPCR)

  • Consider species-specific optimization for cross-species studies

  • Evaluate sample processing time as SH3BGRL2 may have limited stability

What methodological approaches enable accurate quantification of SH3BGRL2 in complex samples?

Precise quantification requires:

• Protein extraction optimization:

  • Buffer selection: PBS with protease inhibitors and appropriate detergents

  • Tissue homogenization protocol standardization

  • Subcellular fractionation for compartment-specific analysis

• Quantitative Western blot strategies:

  • Use recombinant SH3BGRL2 protein standard curves

  • Apply fluorescence-based detection for wider linear range

  • Normalize to total protein rather than single housekeeping proteins

  • Include technical and biological replicates (minimum n=3)

• Advanced quantification methods:

  • Quantitative ELISA with validated sandwich antibody pairs

  • Mass spectrometry with isotope-labeled standards

  • Digital pathology image analysis for IHC quantification

  • Develop and validate digital scoring systems for clinical samples

• Data analysis considerations:

  • Account for technical variation through appropriate statistical methods

  • Apply non-parametric tests for small sample sizes

  • Consider protein degradation during sample storage

How should researchers design experiments to investigate SH3BGRL2's role in signaling pathways?

Signal transduction studies require sophisticated experimental design:

• Pathway stimulation protocols:

  • TGF-β1 pathway: Treat cells with recombinant TGF-β1 with time-course analysis

  • Inhibitor studies: Apply SB431542 (TGF-β receptor inhibitor) and SIS3 (Smad3 inhibitor)

  • Protein turnover: Cycloheximide (CHX) chase experiments to determine stability

  • Proteasomal degradation: MG-132 treatment to assess regulation

• Transcriptional regulation analysis:

  • Chromatin immunoprecipitation to identify direct binding to regulatory regions

  • Luciferase reporter assays with wild-type and mutant promoter constructs

  • siRNA-mediated knockdown of pathway components (SMAD2, SMAD3, SMAD4, TGFBR1, TGFBR2)

• Protein interaction mapping:

  • Co-immunoprecipitation followed by Western blot or proteomics analysis

  • Proximity ligation assays for in situ detection of protein interactions

  • Fluorescence resonance energy transfer (FRET) for live-cell interaction studies

• Functional outcome measurements:

  • Gene expression analysis of downstream targets

  • Phenotypic assays: migration, invasion, proliferation

  • In vivo models to validate pathway connections in physiological context

What key protocols enable successful immunoprecipitation of SH3BGRL2 and its interaction partners?

Effective immunoprecipitation requires:

• Optimized lysis conditions:

  • Buffer composition: Consider RIPA or NP-40 buffer with protease/phosphatase inhibitors

  • Cell density: Harvest cells at 80-90% confluence for optimal protein expression

  • Lysis time: Minimize to prevent degradation of interaction complexes

• IP strategy selection:

  • For tagged SH3BGRL2: Anti-Flag magnetic beads for Flag-SH3BGRL2 constructs

  • For endogenous protein: Validated SH3BGRL2 antibodies pre-conjugated to protein A/G beads

  • Cross-linking considerations: Evaluate whether reversible cross-linking improves complex stability

• Washing protocol optimization:

  • Stringency balance: Sufficient to reduce background without disrupting interactions

  • Multiple wash steps (typically three) with consistent buffer composition

  • Temperature considerations: Perform at 4°C to maintain complex stability

• Detection and analysis:

  • Western blot with antibodies against suspected interaction partners

  • Mass spectrometry for unbiased identification of novel interactions

  • Controls: IgG control IP, input sample (5-10%), and IP supernatant

How can researchers evaluate the expression patterns of SH3BGRL2 across different tissues and cell types?

Comprehensive expression profiling requires:

• Multi-omics approach:

  • Transcriptional analysis: qPCR, RNA-sequencing, microarray data

  • Protein detection: Western blot, IHC, immunofluorescence

  • Database integration: TCGA, CPTAC, GEO, Human Protein Atlas

• Tissue microarray analysis:

  • Design arrays with multiple tissue types and cancer grades

  • Apply standardized IHC protocols (1:20-1:200 dilution)

  • Develop consistent scoring systems for comparative analysis

  • Include normal tissue controls for baseline expression

• Single-cell analysis techniques:

  • Single-cell RNA-sequencing for heterogeneity assessment

  • Multicolor immunofluorescence for co-expression studies

  • Flow cytometry for quantitative cellular distribution

• Developmental and pathological considerations:

  • Analyze expression across developmental stages

  • Compare primary tumors with metastatic sites

  • Correlate with clinical variables (stage, grade, outcome)

What methods should be used to evaluate antibody specificity for SH3BGRL2 in research applications?

Comprehensive validation requires multiple approaches:

• Basic validation methods:

  • Western blot band size confirmation (12-17 kDa observed molecular weight)

  • Peptide competition assays to demonstrate specific binding

  • siRNA/shRNA knockdown to confirm signal reduction

  • Testing in multiple validated positive tissues (placenta, liver, brain)

• Advanced validation strategies:

  • Immunoprecipitation followed by mass spectrometry

  • Comparison of multiple antibodies targeting different epitopes

  • Recombinant protein controls with concentration gradients

  • Cross-reactivity testing against other SH3BGR family members

• Application-specific validation:

  • For IHC: Test on known positive and negative tissues with appropriate controls

  • For WB: Compare with recombinant protein standard and knockout/knockdown samples

  • For IP: Verify pull-down of endogenous protein by mass spectrometry

  • For IF: Co-localization with organelle markers to confirm distribution pattern

• Documentation and reporting:

  • Record lot-specific validation data

  • Document optimization parameters for each application

  • Include comprehensive controls in publications

How do post-translational modifications affect SH3BGRL2 detection and what methodologies address these challenges?

Post-translational modifications present specific challenges:

• Common modifications affecting SH3BGRL2 detection:

  • Phosphorylation: May alter antibody epitope recognition

  • Ubiquitination: Can affect protein stability and turnover

  • Glycosylation: Potentially changes apparent molecular weight

  • Protein-protein interactions: May mask antibody binding sites

• Detection strategies:

  • Phosphatase treatment of samples to identify phosphorylation contributions

  • MG-132 proteasome inhibitor to assess degradation pathways

  • Cycloheximide chase experiments to measure protein stability

  • Denaturation conditions optimization for epitope exposure

• Advanced analytical approaches:

  • 2D gel electrophoresis to separate modified protein forms

  • Mass spectrometry to identify specific modification sites

  • Phospho-specific or modification-specific antibodies when available

  • Native versus reducing conditions to assess structural impacts

• Experimental design considerations:

  • Include appropriate positive controls for each modification

  • Document effective lysis and sample preparation conditions

  • Consider timing of collection for dynamic modifications

  • Record and report all treatment conditions that may affect modifications

How can SH3BGRL2 antibodies be employed to investigate its role in cancer progression across different models?

Multi-model investigation approaches include:

• Cell line model applications:

  • Expression profiling across cancer cell line panels with validated antibodies

  • Functional studies with stable overexpression or knockdown systems

  • Pathway analysis using stimulation/inhibition experiments

  • 3D culture models to assess invasion and morphology changes

• Patient-derived models:

  • Patient-derived xenografts (PDX) with IHC analysis (1:20-1:200 dilution)

  • Primary cell cultures with Western blot detection (1:500-1:5000)

  • Organoid models with immunofluorescence analysis

  • Ex vivo tissue slice cultures for short-term manipulation

• In vivo model considerations:

  • Transgenic models with modulated SH3BGRL2 expression

  • Orthotopic injection models for metastasis studies

  • Tissue-specific conditional knockout approaches

  • Treatment response models with targeted therapies

• Translational research applications:

  • Correlative studies between SH3BGRL2 expression and treatment outcomes

  • Biomarker development for patient stratification

  • Combination studies with standard-of-care therapies

What experimental protocols enable investigation of SH3BGRL2's dual functions in tumor growth versus metastasis?

Specialized protocols for dissecting dual functions:

• Growth inhibition assessment:

  • Proliferation assays: Cell counting, MTT/MTS, colony formation

  • Cell cycle analysis: Flow cytometry with propidium iodide staining

  • Apoptosis quantification: Annexin V/PI, TUNEL assay

  • In vivo tumor growth models with volume measurement

• Metastasis promotion investigation:

  • Migration assays: Wound healing, transwell migration

  • Invasion assays: Matrigel-coated transwell systems

  • 3D invasion models in collagen matrices

  • In vivo metastasis models with lung colonization quantification

• Mechanistic dissection approaches:

  • Spectrin (SPTAN1, SPTBN1) interaction studies via co-IP and functional rescue

  • TGF-β1 pathway activation analysis with inhibitors (SB431542, SIS3)

  • Hippo/TEAD1-Twist1 signaling assessment in different contexts

  • Cytoskeletal reorganization visualization with immunofluorescence

• Context-dependent regulation:

  • Microenvironment co-culture models (tumor-stroma interactions)

  • Extracellular matrix component variation experiments

  • Hypoxia and nutrient deprivation studies

  • Epithelial-mesenchymal transition marker correlation analysis

What methodological approaches best address the challenges of studying SH3BGRL2 in primary patient samples?

Working with clinical samples requires specialized approaches:

• Sample collection and processing optimization:

  • Rapid fixation protocols to preserve protein integrity

  • Standardized processing times for consistency

  • Matched tumor-normal paired sampling when possible

  • Multiple sampling from heterogeneous tumors

• Detection protocol refinement:

  • IHC optimization for different tissue types (1:20-1:200 dilution)

  • Antigen retrieval method comparison (heat-induced vs. enzymatic)

  • Multi-antibody validation approach using different epitopes

  • Digital pathology quantification with standardized scoring systems

• Expression correlation strategies:

  • Multi-level analysis (mRNA by qPCR, protein by WB/IHC)

  • Correlation with clinical parameters (stage, grade, metastasis)

  • Survival analysis with appropriate statistical methods

  • Integration with genomic and proteomic datasets

• Tissue microarray development:

  • Design arrays with multiple tumor regions and matched normal tissue

  • Include progressive disease stages when available

  • Incorporate treatment history information

  • Apply standardized staining and scoring protocols

How might developing technologies enhance the detection and functional analysis of SH3BGRL2?

Emerging methodologies offer new research opportunities:

• Advanced antibody technologies:

  • Single-domain antibodies for improved tissue penetration

  • Proximity-based labeling with antibody-enzyme fusions

  • Intrabodies for live-cell tracking of endogenous protein

  • Nanobodies for super-resolution microscopy applications

• Spatial biology approaches:

  • Spatial transcriptomics to correlate with protein localization

  • Multiplexed ion beam imaging (MIBI) for multi-protein detection

  • Digital spatial profiling for region-specific quantification

  • Single-cell spatial proteomics with subcellular resolution

• Functional genomics integration:

  • CRISPR-Cas9 screening for SH3BGRL2 pathway components

  • CRISPR activation/inhibition for endogenous modulation

  • Base editing for specific mutation introduction

  • Prime editing for precise genetic modifications

• Computational biology applications:

  • Machine learning for expression pattern recognition

  • Protein structure prediction with AlphaFold for interaction modeling

  • Systems biology approaches for pathway integration

  • Patient stratification algorithms based on expression patterns

What are the critical considerations when developing experimental systems to test SH3BGRL2-targeting therapeutics?

Therapeutic development requires specialized experimental design:

• Target validation approaches:

  • Genetic modulation: CRISPR, shRNA, overexpression in multiple models

  • Patient-derived systems to confirm clinical relevance

  • Careful controls for off-target effects

  • Context-dependent function assessment (growth vs. metastasis)

• Assay development considerations:

  • Physiologically relevant readouts for dual function assessment

  • High-throughput compatible systems for screening

  • Translation between in vitro and in vivo models

  • Biomarker development for response prediction

• Therapeutic strategy evaluation:

  • Direct targeting vs. pathway modulation approaches

  • Context-specific intervention timing (primary vs. metastatic disease)

  • Combination strategies with standard therapies

  • Resistance mechanism anticipation and monitoring

• Translational research planning:

  • Biomarker-driven patient selection strategies

  • Pharmacodynamic marker development

  • Safety assessment in multiple tissues given widespread expression

  • Clinical trial design considerations for context-dependent effects