SCLY Antibody

What is SCLY and what role does it play in selenium metabolism?

SCLY (Selenocysteine Lyase) is an enzyme that catalyzes the decomposition of L-selenocysteine to L-alanine and elemental selenium . It plays a critical role in the recycling of selenocysteine from selenoproteins, releasing selenium for reuse in the biosynthesis of new selenoproteins . SCLY is primarily active in the liver and kidneys, contributing to maintaining selenium homeostasis, which is vital for numerous biological functions including antioxidant defense and thyroid hormone metabolism . The enzyme belongs to the class-V pyridoxal-phosphate-dependent aminotransferase family and forms homodimers in mouse liver .

What is the molecular weight and cellular localization of SCLY?

The calculated molecular weight of human SCLY is approximately 49 kDa, with the observed molecular weight in Western blots being approximately 48 kDa . The protein is primarily localized in the cytoplasm . According to experimental data from multiple antibody validation studies, SCLY protein exhibits consistent molecular weight detection across various cell lines and tissue samples .

What applications are SCLY antibodies validated for in research settings?

SCLY antibodies have been validated for the following applications:

Western blot is the most common and widely validated application for SCLY antibodies . Immunohistochemistry has been validated for some antibodies, particularly in human breast cancer tissue and other samples with appropriate antigen retrieval methods .

What species reactivity is available for commercial SCLY antibodies?

Commercial SCLY antibodies show diverse species reactivity profiles:

When working with species not listed as validated, researchers should consider conducting a BLAST comparison between the target species and the immunogen sequence to assess potential cross-reactivity . Pilot testing is recommended for non-validated species applications.

What cell lines and tissues serve as positive controls for SCLY antibody validation?

Based on antibody validation data, the following cell lines and tissues serve as reliable positive controls for SCLY detection:

These samples have demonstrated consistent SCLY expression in Western blot and immunohistochemistry applications . High expression levels have been reported in liver, kidney, and testis tissues, making these optimal positive control samples for antibody validation .

What methodologies are recommended for validating SCLY antibody specificity?

A comprehensive SCLY antibody validation strategy should incorporate multiple approaches:

• Multiple antibody comparison: Use antibodies targeting different epitopes of SCLY (e.g., C-terminal vs. N-terminal) to confirm detection patterns. Abcam ab190983 targets the C-terminal region , while other antibodies may target N-terminal regions .

• Positive control selection: Include tissues with known high expression (liver, kidney, testis) and cell lines with confirmed expression (U87, HeLa, 293T) .

• Knockout/knockdown validation: Generate SCLY knockouts or knockdowns to verify antibody specificity. The antibody signal should be absent or significantly reduced in these samples.

• Western blot optimization: For human samples, expected band size is approximately 48 kDa . Multiple bands may indicate splice variants or post-translational modifications.

• Cross-reactivity assessment: When testing in non-validated species, conduct sequence homology analysis of the immunogen peptide with the target species .

How can researchers optimize Western blot protocols for consistent SCLY detection?

Based on experimental validation data from multiple sources, the following protocol optimizations are recommended:

Sample Preparation:

• For tissue samples: Homogenize in RIPA buffer (15 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.25% sodium deoxycholate), sonicate, and centrifuge at 16,000 × g for 5 minutes .

• Protein loading: 5-40 μg total protein per lane is sufficient for detection .

Electrophoresis and Transfer:

• Use 4-20% SDS-PAGE gels for optimal separation .

• Transfer to PVDF membranes (e.g., Immobilon-FL) .

Antibody Incubation:

• Primary antibody dilutions:

  • For Proteintech 67606-1-Ig: 1:1000-1:4000

  • For Proteintech 10667-1-AP: 1:500-1:2000

  • For Boster PA1995: 0.1-0.5 μg/ml

• Incubation time: 1.5 hours at room temperature or overnight at 4°C .

Detection:

• Infrared imaging systems (e.g., Odyssey Infrared Imager) provide sensitive and quantitative detection .

• Expected band size is approximately 48 kDa .

What are the implications of altered SCLY expression in metabolic and oxidative stress disorders?

Research has revealed significant insights into SCLY's role in metabolic disorders:

• Non-alcoholic fatty liver disease (NAFLD): SCLY expression is reduced in non-alcoholic steatohepatitis (NASH) compared to healthy controls . This suggests a potential disruption in selenium metabolism in the progression of liver disease.

• Glucose and lipid metabolism: Disruption of Scly in mouse models affects glucose and lipid homeostasis . Specifically, Scly disruption increases levels of PTP1B, an insulin-signaling inhibitor, suggesting a mechanistic link between selenium metabolism and insulin resistance.

• Oxidative stress protection: SCLY's role in selenium recycling is essential for maintaining adequate levels of selenoproteins, which function as antioxidants . Impaired SCLY function may contribute to increased oxidative damage in tissues.

• Tissue-specific effects: While SCLY is expressed in all mouse tissues examined, the highest expression is in liver, kidney, and testis , suggesting tissue-specific roles in selenium metabolism that may differently impact disease progression in these organs.

These findings highlight the importance of SCLY in selenium homeostasis and its potential as a therapeutic target in metabolic disorders characterized by oxidative stress and insulin resistance.

How can deep learning approaches be applied to develop improved antibodies for targets like SCLY?

Recent advances in deep learning for antibody development offer promising approaches for generating SCLY-targeting antibodies with enhanced specificity and developability:

• Generative Adversarial Networks (GANs): GANs can generate novel antibody sequences with desirable developability attributes . The Wasserstein GAN with Gradient Penalty has been used to maintain diversity while keeping sequences within specified boundary conditions.

• Key developability attributes to optimize:

  • Medicine-likeness (resemblance to marketed antibody biotherapeutics)

  • High humanness (>90% recommended)

  • Absence of unpaired cysteine residues

  • Absence of N-linked glycosylation motifs

  • No chemical liabilities in CDRs

• Experimental validation pipeline: In-silico generated antibodies should undergo rigorous experimental validation including:

  • Expression level assessment

  • Monomer content analysis

  • Thermal stability testing

  • Hydrophobicity evaluation

  • Self-association testing

  • Non-specific binding assessment

• Performance metrics: In one study, deep learning-generated antibodies showed high expression, monomer content, and thermal stability along with low hydrophobicity, self-association, and non-specific binding when produced as full-length monoclonal antibodies .

This approach could potentially accelerate the development of highly specific SCLY antibodies with improved performance characteristics for research and potential therapeutic applications.

What considerations are important when selecting paired heavy and light chains for SCLY antibody development?

Research on antibody pairing strategies reveals important considerations for SCLY antibody development:

• Native vs. random pairing: Studies comparing natively paired versus randomly paired antibody libraries found that antibodies with native light chains were more likely to bind their target than antibodies with non-native light chains . This suggests a higher false positive rate for antibodies from randomly paired libraries.

• Impact on binding properties: Different light chains paired with the same heavy chain can lead to highly divergent binding properties, and minor changes can significantly affect specificity and affinity .

• Library screening efficiency: Natively paired libraries showed advantages in both sensitivity and specificity for antibody discovery programs . The randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate.

• Validation requirements: When developing SCLY antibodies, researchers should consider:

  • Confirmation of native heavy-light chain pairing

  • Validation across multiple binding assays to characterize therapeutic potential

  • Assessment of false positive rates between native and non-native pairing approaches

These findings emphasize the importance of maintaining native heavy and light chain pairing during SCLY antibody development to maximize discovery of high-quality, specific antibodies with optimal binding characteristics.