SH3YL1 Antibody

What is SH3YL1 protein and why is it significant in research?

SH3YL1 (Src homology 3 domain-containing YSC84-like 1) is a highly conserved noncatalytic protein found in yeasts and vertebrates that binds to cytoplasmic tyrosine kinase. It has gained significant research interest as it functions as a regulator of NADPH oxidase 4 (NOX4), which is involved in oxidative stress pathways. SH3YL1 is expressed in various human organs, including the kidney, and has been implicated in oxidative stress-induced inflammatory processes in diabetic conditions . The protein regulates the hair cycle and hair follicle formation in keratinocytes, but its biological functions extend beyond this, making it an important target for various research fields, particularly in renal and diabetic studies .

What are the common applications of SH3YL1 antibodies in research?

SH3YL1 antibodies are valuable tools in multiple research applications:

• Immunoblotting (Western blotting): Used at concentrations of 0.04-0.4 μg/mL to detect SH3YL1 protein expression in tissue lysates

• Immunohistochemistry: Applied at dilutions of 1:50-1:200 to visualize SH3YL1 localization in tissue sections

• Biomarker studies: To measure SH3YL1 protein levels in plasma and urine samples for predicting renal outcomes in diabetic patients

• Protein-protein interaction studies: To investigate how SH3YL1 interacts with other proteins, particularly in oxidative stress pathways

These applications allow researchers to study SH3YL1's role in normal physiology and pathological conditions, especially in diabetes-related kidney disease .

What is the typical immunogen sequence used to generate SH3YL1 antibodies?

Commercial SH3YL1 antibodies are typically generated using a specific immunogen sequence: FTYCKSRGLFAGVSLEGSCLIERKETNRKFYCQDIRAYDILFGDTPRPAQAEDLYEILDSFTEKYENEG . This sequence corresponds to a region of the human SH3YL1 protein that elicits a strong immune response in the host animal (commonly rabbits) and ensures specificity of the resulting antibody. When selecting an SH3YL1 antibody for cross-species applications, it's important to note that this sequence has high homology across species - approximately 93% identity with mouse and rat orthologs, making many commercial antibodies suitable for studies across these species .

How should SH3YL1 antibodies be stored and handled to maintain optimal activity?

For optimal performance and longevity of SH3YL1 antibodies:

• Storage temperature: Store at -20°C for long-term preservation

• Shipping conditions: Antibodies are typically shipped on wet ice to maintain stability

• Working aliquots: To prevent freeze-thaw cycles, prepare small working aliquots for regular use

• Buffer conditions: Most commercial preparations come in buffered aqueous glycerol solutions, which help maintain antibody stability

• Handling precautions: Avoid repeated freeze-thaw cycles, exposure to high temperatures, and contamination

Following these practices will help maintain antibody specificity and sensitivity throughout your experimental timeline, ensuring consistent and reliable results in immunoassays.

What are the recommended protocols for using SH3YL1 antibodies in immunohistochemistry?

For effective immunohistochemical staining with SH3YL1 antibodies:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Embed in paraffin and section at 4-6 μm thickness

  • Deparaffinize and rehydrate sections through graded alcohols

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Maintain at 95-98°C for 15-20 minutes, then cool to room temperature

• Antibody incubation:

  • Block with appropriate serum (5% normal goat serum) for 1 hour

  • Dilute primary SH3YL1 antibody 1:50-1:200 in blocking buffer

  • Incubate overnight at 4°C in a humidified chamber

• Detection:

  • Use appropriate secondary antibody system (HRP/DAB or fluorescent-tagged)

  • Counterstain with hematoxylin for brightfield or DAPI for fluorescence

  • Mount with appropriate medium

For optimal staining, it's recommended to include positive and negative controls to validate specificity and determine optimal antibody dilution for your specific tissue type.

What validation steps should be performed when using a new SH3YL1 antibody?

Comprehensive validation of a new SH3YL1 antibody should include:

• Western blot analysis:

  • Verify single band at expected molecular weight (approximately 26-28 kDa)

  • Test multiple tissue/cell lysates with known SH3YL1 expression levels

  • Include positive and negative control samples

• Specificity testing:

  • Pre-absorption with immunizing peptide to confirm specific binding

  • Testing in SH3YL1 knockout/knockdown cells or tissues

  • Cross-reactivity assessment with related proteins

• Application-specific validation:

  • For IHC: Compare staining patterns with published literature

  • For ELISA: Generate standard curves with recombinant protein

  • For IP: Confirm pull-down of SH3YL1 by mass spectrometry

• Lot-to-lot consistency:

  • Compare performance metrics between different antibody lots

  • Document staining intensity, background, and specificity

Thorough validation ensures reliable and reproducible results in your experimental systems, preventing wasted time and resources on potentially non-specific antibodies.

How can SH3YL1 antibodies be used to investigate its role in NADPH oxidase regulation?

To investigate SH3YL1's role in NADPH oxidase regulation:

• Co-immunoprecipitation studies:

  • Use SH3YL1 antibodies to pull down protein complexes

  • Probe for NOX4 and other NADPH oxidase components

  • Identify direct binding partners and regulatory interactions

• Proximity ligation assays:

  • Visualize direct interactions between SH3YL1 and NOX4 in situ

  • Quantify interaction dynamics under various cellular stresses

• Subcellular localization:

  • Perform immunofluorescence with SH3YL1 antibodies

  • Co-stain with NOX4 antibodies and organelle markers

  • Track translocation under oxidative stress conditions

• Functional assays:

  • Measure ROS production in cells with SH3YL1 overexpression or knockdown

  • Compare NOX4 activity in the presence/absence of SH3YL1

  • Correlate protein levels (detected by antibodies) with enzymatic activity

These approaches can help elucidate the molecular mechanisms by which SH3YL1 regulates NOX4 activity and subsequent oxidative stress responses, particularly in diabetic kidney disease contexts .

What is the evidence supporting SH3YL1 as a biomarker for diabetic kidney disease progression?

Recent research has established SH3YL1 as a promising biomarker for diabetic kidney disease progression:

• Clinical correlation studies:

  • Urinary SH3YL1-to-creatinine ratio (USCR) levels show significant negative correlation with estimated glomerular filtration rate (eGFR)

  • USCR shows positive correlation with urinary albumin-to-creatinine ratio (UACR)

  • USCR levels differ significantly across chronic kidney disease (CKD) stages and albuminuria groups

• Predictive value:

  • Patients in the highest tertile of USCR showed significantly lower probability of renal event-free survival over 3 years

  • After full adjustment for confounding factors, the highest USCR tertile had an adjusted hazard ratio of 4.636 (95% CI: 1.416–15.181, p=0.011) for renal events

• Biological plausibility:

  • SH3YL1 is produced by renal cells (podocytes, mesangial cells, proximal tubule cells) in response to high glucose stimulation

  • It's associated with oxidative stress-induced inflammatory processes in diabetic conditions

  • As a regulator of NOX4, SH3YL1 has direct mechanistic links to renal oxidative stress

This evidence suggests that SH3YL1 antibody-based assays could provide clinically valuable information for risk stratification in diabetic patients, potentially enabling earlier intervention in high-risk individuals.

How does SH3YL1 participate in EGFR sorting and degradation pathways?

SH3YL1 plays a significant role in epidermal growth factor receptor (EGFR) trafficking and degradation:

• Endosomal sorting:

  • SH3YL1 cooperates with ESCRT-I (Endosomal Sorting Complex Required for Transport-I) in the sorting of EGFR

  • It facilitates the delivery of ubiquitinated EGFR from early endosomes to multivesicular bodies (MVBs)

• Intralumenal vesicle formation:

  • SH3YL1 contributes to the regulatory mechanism underlying formation of intralumenal vesicles

  • This process is crucial for the generation of MVBs and subsequent lysosomal degradation of EGFR

• Degradation pathway:

  • Through its interaction with the ESCRT machinery, SH3YL1 helps direct EGFR to lysosomes for degradation

  • This represents a critical mechanism for downregulating EGFR signaling after receptor activation

Understanding these pathways is important for cancer research, as dysregulation of EGFR trafficking and degradation can contribute to sustained EGFR signaling and potentially promote tumor growth and progression. SH3YL1 antibodies are valuable tools for studying these complex cellular processes through techniques like immunoprecipitation and immunofluorescence microscopy.

What are common issues encountered when using SH3YL1 antibodies in Western blotting and how can they be resolved?

Common issues with SH3YL1 antibodies in Western blotting include:

• Weak or no signal:

  • Solution: Optimize antibody concentration (try range of 0.04-0.4 μg/mL as recommended)

  • Increase protein loading or use more sensitive detection systems

  • Verify sample preparation to ensure protein integrity

  • Test different antigen retrieval or blocking methods

• Multiple bands or non-specific binding:

  • Solution: Increase stringency of washes (higher salt concentration, longer washing)

  • Optimize blocking conditions (try different blocking agents)

  • Reduce primary antibody concentration

  • Pre-absorb antibody with immunizing peptide to confirm specificity

• Inconsistent results:

  • Solution: Standardize lysate preparation and protein quantification

  • Use loading controls consistently

  • Document exact protocol parameters for reproducibility

  • Consider the effects of post-translational modifications on migration pattern

• High background:

  • Solution: Increase blocking time or BSA/milk concentration

  • Use fresher membrane blocking reagents

  • Ensure thorough washing between steps

  • Reduce antibody concentration or incubation time

For optimal results with SH3YL1 antibodies in Western blotting, it's recommended to start with the manufacturer's suggested protocol and systematically optimize each parameter based on your specific experimental system.

How do you determine the optimal antibody concentration for SH3YL1 ELISA assays in biomarker studies?

To determine the optimal antibody concentration for SH3YL1 ELISA assays:

• Antibody titration:

  • Perform checkerboard titration with varying concentrations of capture and detection antibodies

  • Test ranges from 0.1-10 μg/mL for capture antibody

  • For detection antibody, test dilutions from 1:100 to 1:10,000

  • Identify combination that gives highest signal-to-noise ratio

• Standard curve optimization:

  • Use recombinant SH3YL1 protein to generate standard curves

  • Test different concentration ranges (e.g., 0-5000 pg/mL)

  • Ensure linear response across clinically relevant concentrations

  • Verify lower limit of detection and quantification

• Sample matrix effects:

  • Test antibody performance in the specific biological matrix (plasma, urine)

  • Determine if sample dilution is necessary to minimize matrix interference

  • Spike known concentrations of recombinant protein to assess recovery

• Validation with clinical samples:

  • Compare results with established reference ranges or correlate with clinical parameters

  • Assess intra-assay and inter-assay precision

  • Determine stability of samples under various storage conditions

For biomarker studies like those measuring urinary or plasma SH3YL1 in diabetic patients, assay optimization is critical to ensure accurate quantification across the wide range of concentrations observed in clinical samples (reported median values: 7.73 pg/mgCr for USCR and 301.1 pg/mL for plasma SH3YL1) .

What controls should be included when studying SH3YL1 expression in different tissue types?

When studying SH3YL1 expression across different tissues, include these controls:

• Positive tissue controls:

  • Tissues with known high SH3YL1 expression (kidney, skin)

  • Recombinant expression systems overexpressing SH3YL1

  • Cell lines with validated endogenous expression

• Negative controls:

  • Antibody diluent only (no primary antibody)

  • Isotype control antibody at same concentration

  • Pre-immune serum from the same species

  • SH3YL1 knockout tissues or cells (if available)

• Specificity controls:

  • Pre-absorption with immunizing peptide

  • Secondary antibody only controls

  • Competitive blocking with recombinant SH3YL1

• Technical controls:

  • Housekeeping protein detection (loading controls)

  • Tissue-specific markers to confirm tissue identity

  • RNA expression correlation (RT-PCR or RNA-seq data)

  • Multiple antibodies targeting different epitopes of SH3YL1

• Reference standards:

  • Include standardized positive samples in each experiment

  • Use calibrated protein standards for quantitative comparisons

  • Compare results with Human Protein Atlas data for tissue expression patterns

Proper controls ensure that observed expression patterns are genuine and not artifacts of the detection method, particularly important when comparing SH3YL1 expression across different tissues or disease states.

How can SH3YL1 antibodies be used to investigate oxidative stress in diabetic nephropathy models?

SH3YL1 antibodies can be valuable tools for investigating oxidative stress in diabetic nephropathy through several approaches:

• Expression analysis in disease progression:

  • Immunohistochemical staining of kidney sections from diabetic animal models

  • Quantification of SH3YL1 expression changes during disease progression

  • Correlation with markers of oxidative damage and NOX4 expression

• Mechanistic studies:

  • Co-localization studies with NOX4 and oxidative stress markers

  • Proximity ligation assays to visualize direct SH3YL1-NOX4 interactions

  • Immunoprecipitation to identify protein complexes formed under diabetic conditions

• Intervention studies:

  • Monitor SH3YL1 expression changes in response to antioxidant therapies

  • Track protein levels following NOX inhibitor treatment

  • Correlate with improvements in renal function and oxidative stress parameters

• Biofluid analysis:

  • Develop antibody-based ELISA assays to measure urinary and plasma SH3YL1 levels

  • Correlate with disease severity and progression metrics

  • Use as pharmacodynamic markers in intervention studies

These approaches can help elucidate the role of SH3YL1 in oxidative stress-mediated kidney damage in diabetes and potentially identify new therapeutic targets or biomarkers for diabetic nephropathy .

What are the implications of SH3YL1's role in cell signaling for cancer research?

SH3YL1's involvement in cell signaling pathways has several implications for cancer research:

• EGFR trafficking and degradation:

  • SH3YL1 cooperates with ESCRT-I in sorting and degradation of EGFR

  • Dysregulation could lead to prolonged EGFR signaling, a hallmark of many cancers

  • Potential therapeutic target for cancers with EGFR-dependent growth

• Oxidative stress regulation:

  • As a regulator of NOX4, SH3YL1 impacts cellular redox balance

  • Cancer cells often exhibit altered redox states

  • Targeting this pathway could affect cancer cell vulnerability to oxidative stress

• Cytoskeletal regulation:

  • SH3YL1 interacts with cytoplasmic tyrosine kinases

  • May influence cell migration, invasion, and metastatic potential

  • Could contribute to tumor cell plasticity and adaptability

• Biomarker potential:

  • Expression patterns may correlate with tumor aggressiveness or therapeutic response

  • Could serve as a prognostic or predictive biomarker in specific cancer types

  • May help stratify patients for targeted therapies

SH3YL1 antibodies can be used to investigate these aspects through techniques like tissue microarray analysis, immunohistochemical profiling of tumor samples, and functional studies in cancer cell lines. Understanding SH3YL1's contribution to cancer biology could potentially reveal new therapeutic vulnerabilities or diagnostic opportunities.

How do you design experiments to study the relationship between SH3YL1 and NOX4 in oxidative stress conditions?

To investigate the relationship between SH3YL1 and NOX4 in oxidative stress conditions:

• Expression correlation studies:

  • Use SH3YL1 and NOX4 antibodies for dual immunostaining in tissues

  • Quantify protein levels by Western blot across oxidative stress conditions

  • Measure mRNA and protein expression in response to oxidative stressors

  • Example design: Treat renal cells with high glucose (25mM) for 24-72 hours, then compare SH3YL1 and NOX4 protein/mRNA levels

• Functional interaction studies:

  • Perform co-immunoprecipitation using SH3YL1 antibodies under normal vs. oxidative stress

  • Utilize proximity ligation assays to visualize and quantify interactions

  • Conduct FRET or BRET assays to assess direct protein interactions

  • Example design: Immunoprecipitate SH3YL1 from H₂O₂-treated vs. untreated cells, then probe for NOX4

• Knockdown/overexpression approaches:

  • Manipulate SH3YL1 levels and measure effects on NOX4 activity

  • Assess ROS production using fluorescent probes (DCF-DA, MitoSOX)

  • Determine effects on downstream oxidative damage markers

  • Example design: Create stable SH3YL1 knockdown cell lines, measure NOX4 activity and ROS production

• In vivo models:

  • Generate conditional SH3YL1 knockout mice

  • Induce diabetic conditions and measure renal NOX4 activity

  • Assess oxidative stress parameters and kidney function

  • Example design: Compare diabetic wild-type and SH3YL1 knockout mice for renal NOX4 expression, activity, and oxidative damage

These experimental approaches would provide comprehensive insights into how SH3YL1 regulates NOX4 and contributes to oxidative stress, particularly in the context of diabetic nephropathy .

What emerging technologies might enhance the utility of SH3YL1 antibodies in research?

Several emerging technologies promise to enhance SH3YL1 antibody applications:

• Antibody engineering:

  • Development of single-domain antibodies or nanobodies against SH3YL1

  • Site-specific conjugation of fluorophores or enzymes for improved sensitivity

  • Generation of bispecific antibodies targeting SH3YL1 and interacting partners

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize SH3YL1 localization at nanoscale resolution

  • Live-cell imaging with genetically encoded anti-SH3YL1 intrabodies

  • Expansion microscopy to physically enlarge specimens for detailed protein localization

• Proteomics integration:

  • Antibody-based proximity labeling (BioID, APEX) to map SH3YL1 interactome

  • Single-cell proteomics with antibody-based detection of SH3YL1

  • Mass cytometry (CyTOF) for multiplexed protein detection including SH3YL1

• Diagnostic applications:

  • Development of point-of-care lateral flow assays for SH3YL1 detection in urine

  • Microfluidic-based assays for rapid quantification in clinical samples

  • Digital ELISA platforms for ultrasensitive detection of low-abundance SH3YL1

These technologies could significantly enhance our ability to study SH3YL1's function, localization, and interactions in both research and clinical contexts, potentially leading to improved diagnostic tools for diseases like diabetic nephropathy where SH3YL1 has demonstrated biomarker potential .

What are the key research questions regarding SH3YL1 that remain unanswered?

Several critical research questions about SH3YL1 remain to be addressed:

• Regulatory mechanisms:

  • What factors regulate SH3YL1 expression in different tissues?

  • How is SH3YL1 activity modulated post-translationally?

  • What signaling pathways control SH3YL1 function?

• Structural biology:

  • What is the three-dimensional structure of SH3YL1?

  • How does SH3YL1 physically interact with NOX4 and ESCRT components?

  • What conformational changes occur during activation?

• Physiological roles:

  • What is the complete tissue expression profile of SH3YL1 beyond currently known locations?

  • What are the phenotypic consequences of SH3YL1 deficiency in different organs?

  • How does SH3YL1 function differ across species?

• Pathological implications:

  • Does SH3YL1 play roles in diseases beyond diabetic nephropathy?

  • Could SH3YL1 be a therapeutic target for oxidative stress-related disorders?

  • What is the prognostic value of SH3YL1 as a biomarker in various conditions?

• Mechanistic questions:

  • How exactly does SH3YL1 regulate NOX4 activity at the molecular level?

  • What is the relationship between SH3YL1's roles in EGFR trafficking and oxidative stress?

  • Does SH3YL1 interact with other NADPH oxidase family members?

Addressing these questions will require diverse approaches including knockout models, structural studies, and comprehensive clinical investigations using well-characterized SH3YL1 antibodies.

How might SH3YL1 antibody research contribute to personalized medicine approaches for diabetic nephropathy?

SH3YL1 antibody-based research could significantly advance personalized medicine for diabetic nephropathy through:

• Risk stratification:

  • Development of clinical-grade ELISA assays for urinary and plasma SH3YL1 measurement

  • Identification of SH3YL1 level thresholds that predict rapid disease progression

  • Integration with other biomarkers to create risk prediction algorithms

  • Example: Patients in the highest tertile of USCR had 4.636 times higher risk of renal events

• Treatment response monitoring:

  • Use of SH3YL1 levels as pharmacodynamic biomarkers

  • Longitudinal monitoring to assess therapeutic efficacy

  • Earlier identification of non-responders for treatment modification

• Novel therapeutic targets:

  • Development of therapies targeting SH3YL1-NOX4 interaction

  • Screening for small molecules that modulate SH3YL1 function

  • Potential for pathway-specific antioxidant approaches

• Companion diagnostics:

  • SH3YL1 antibody-based assays as companion diagnostics for NOX inhibitors

  • Patient selection for clinical trials based on SH3YL1 expression patterns

  • Treatment decisions guided by SH3YL1 pathway activation status

The development of standardized, validated SH3YL1 antibody-based diagnostic tests could enable earlier intervention in high-risk patients, therapy selection based on molecular mechanism, and more precise monitoring of disease progression and treatment response. This personalized approach could significantly improve outcomes in diabetic nephropathy, which remains a leading cause of end-stage renal disease worldwide .