SLC7A2 Antibody, Biotin conjugated

What is SLC7A2 and what cellular functions does it serve?

SLC7A2 (Solute Carrier Family 7 Member 2) functions as a permease involved in the transport of cationic amino acids, including L-arginine, L-lysine, L-ornithine, and L-homoarginine. The protein exists in different isoforms created by alternative splicing, each with varying substrate affinities . Isoform 1 functions as a permease with much higher affinity for L-arginine compared to isoform 2, which operates as a low-affinity, high-capacity transporter .

SLC7A2 plays a critical role in arginine metabolism, which is essential for various physiological processes including cell division, proliferation, wound healing, and immune functions. Importantly, SLC7A2 serves as an important regulator of innate and adaptive immunity in macrophages by increasing arginine transport . Recent research has also identified SLC7A2 as highly expressed in astrocytes, vascular cells, and leptomeningeal cells in the brain, suggesting tissue-specific functions .

What are the key applications for SLC7A2 antibodies in research?

SLC7A2 antibodies have several important research applications:

• Western Blotting (WB): Used to detect and quantify SLC7A2 protein expression in tissue or cell lysates. The observed molecular weight of SLC7A2 is approximately 100 kDa, though the calculated molecular weight is around 49.7 kDa . This discrepancy may be due to post-translational modifications.

• Immunohistochemistry (IHC): Used to localize SLC7A2 protein in tissue sections. Most antibodies against SLC7A2 are suitable for IHC-P (paraffin-embedded sections) .

• ELISA: Biotin-conjugated antibodies are particularly useful for ELISA applications, with recommended dilutions typically ranging from 1:500 to 1:1000 .

• Neuroinflammation Studies: SLC7A2 antibodies can be used to investigate the role of this transporter in neuroinflammatory processes, particularly in conditions like Huntington's disease where SLC7A2 expression is upregulated .

What is the significance of biotin conjugation for SLC7A2 antibodies?

Biotin conjugation of SLC7A2 antibodies provides several methodological advantages:

• Signal Amplification: The biotin-avidin/streptavidin system offers one of the strongest non-covalent biological interactions known, with a dissociation constant (Kd) of approximately 10^-15 M. This allows for significant signal amplification in detection systems.

• Versatility in Detection Systems: Biotin-conjugated antibodies can be detected using various avidin/streptavidin conjugated reporter molecules (HRP, fluorophores, gold particles), offering flexibility in experimental design.

• ELISA Application Optimization: For SLC7A2 detection in ELISA, biotin-conjugated antibodies typically demonstrate optimal performance at dilutions of 1:500-1:1000 .

• Multi-color Staining: Biotin-conjugated primary antibodies facilitate multi-color staining protocols by allowing differential detection of multiple targets simultaneously.

The biotin conjugation does not appear to interfere with the antibody's ability to recognize the SLC7A2 protein epitopes, as indicated by successful application in detection systems described in the literature.

How can SLC7A2 antibodies be used to investigate the role of arginine transport in neuroinflammation and Huntington's disease?

Recent research has identified SLC7A2 as one of the most significantly upregulated genes when normal Huntingtin (HTT) is deleted . This finding positions SLC7A2 antibodies as valuable tools for investigating the pathophysiology of Huntington's disease (HD) and related neuroinflammatory processes.

Methodological approaches include:

• Characterizing Expression Patterns: SLC7A2 antibodies can be used to track upregulation of this transporter in HD cellular models and patient samples through immunohistochemistry and western blotting. Research has shown selective upregulation of SLC7A2 in both HD cellular models and patient samples .

• Monitoring Neuroinflammatory Responses: SLC7A2's role in arginine transport makes it a critical mediator of nitric oxide (NO) production. HD cells exhibit an overactive response to neuroinflammatory challenges, demonstrated by abnormally high inducible nitric oxide synthase (iNOS) induction and NO production. Researchers can use SLC7A2 antibodies to characterize this pathway by:

  • Examining co-localization with inflammatory markers

  • Quantifying expression levels in relation to nitrosative stress markers

  • Tracking changes in expression following inflammatory stimuli

• Validating Knockout Studies: Research has shown that knocking out SLC7A2 blocked iNOS induction and NO production in HD cell models (STHdhQ111 cells) . SLC7A2 antibodies can be used to confirm knockout efficiency and correlate loss of protein expression with functional changes.

What experimental considerations should be made when using biotin-conjugated SLC7A2 antibodies?

When designing experiments with biotin-conjugated SLC7A2 antibodies, researchers should consider:

• Endogenous Biotin Interference: Tissues with high endogenous biotin (liver, kidney, brain) may produce background signal. Mitigation strategies include:

  • Pre-blocking with streptavidin/avidin

  • Using biotin blocking kits

  • Including appropriate controls to distinguish specific from non-specific binding

• Epitope Accessibility: The antibody targeting the C-terminal region of SLC7A2 (amino acids 500 to C-terminus) requires consideration of epitope accessibility in fixed tissues . The peptide sequence recognized by some available antibodies includes: "FLAFVLGLSVLTTYGVHAITRLEAWSLALLALFLVLFVAIVLTIWRQPQNQQKVAFMVPFLPFLPAFSILVNIYLMVQLSADTWVRFSIWMAIGFLIYFYGIRHSLEGHLRDENNEEDAYPDNVHAAAEEКСAIQANDHНPRNLSSPFIFHEKTSEF" .

• Isoform Specificity: SLC7A2 exists in different splice variants with distinct functions:

  • Isoform 1: High-affinity transporter for L-arginine

  • Isoform 2: Low-affinity, high-capacity transporter

  Researchers should verify which isoform(s) their antibody recognizes, particularly when investigating isoform-specific functions.

• Application-Specific Optimization:

  • For ELISA: Recommended dilution of 1:500-1:1000

  • For IHC-P: Optimize dilution based on tissue-specific parameters

  • For Western blotting: Based on validation data, the expected molecular weight is approximately 100 kDa despite a calculated weight of ~49.7 kDa

How can researchers validate the specificity of SLC7A2 antibodies in their experimental systems?

Rigorous validation of SLC7A2 antibodies is essential for reliable research outcomes. Recommended validation approaches include:

• Positive and Negative Control Tissues: Based on available data, researchers should include:

  • Positive controls: Placenta, skeletal muscle, and ovary tissues have been validated for SLC7A2 detection

  • Negative controls: Include tissues known to have low SLC7A2 expression or use blocking peptides

• SLC7A2 Knockout Validation: CRISPR-Cas9 knockout of SLC7A2 (as performed in STHdhQ7 and Q111 cells) provides an ideal negative control . Researchers should observe absence of signal in knockout samples when using specific antibodies.

• Western Blot Profile Analysis: Validation by western blot should reveal bands at approximately 100 kDa. Multi-tissue analysis has confirmed reactivity in:

  • Human placenta tissue

  • Rat skeletal muscle and ovary tissues

  • Mouse skeletal muscle tissue

  • Mouse C2C12 cell lysates

• Peptide Competition Assay: Preincubation of the antibody with the immunizing peptide should abolish specific staining. For some commercially available antibodies, this corresponds to the recombinant human CAT2/SLC7A2 protein (Position: M1-F658) .

• Cross-Reactivity Assessment: Most commercially available SLC7A2 antibodies show reactivity across human, mouse, and rat samples, with some also recognizing pig, rabbit, and dog orthologs .

What protocols are recommended for using biotin-conjugated SLC7A2 antibodies in ELISA applications?

For optimal ELISA performance with biotin-conjugated SLC7A2 antibodies:

• Sample Preparation:

  • Cell lysates should be prepared using non-denaturing lysis buffers to preserve the native protein conformation

  • For tissue homogenates, avoid detergents that might interfere with biotin-streptavidin interactions

• Protocol Recommendations:

  • Coating: Use purified recombinant human SLC7A2 protein (604-658AA region has been successfully used as an immunogen)

  • Blocking: 3-5% BSA in PBS is recommended to reduce background

  • Antibody Dilution: 1:500-1:1000 for biotin-conjugated SLC7A2 antibodies

  • Detection: Streptavidin-HRP followed by appropriate substrate

  • Incubation Times: Overnight at 4°C for primary antibody binding generally yields optimal results

• Controls:

  • Include a standard curve using recombinant SLC7A2 protein

  • Include negative controls (samples known not to express SLC7A2)

  • Include isotype controls to assess non-specific binding

How can researchers investigate SLC7A2's role in arginine metabolism and inflammatory responses?

SLC7A2's critical role in arginine transport makes it a key player in inflammatory processes. Methodological approaches for investigating this function include:

• Arginine Transport Studies:

  • Measure uptake of radiolabeled arginine in cells with varying SLC7A2 expression levels

  • Compare transport kinetics between cells expressing different SLC7A2 isoforms

  • Assess arginine transport in the presence of inflammatory stimuli (e.g., LPS, cytokines)

• Nitric Oxide Production Assessment:

  • Use Griess assay to measure nitrite levels as an indicator of NO production

  • Correlate NO production with SLC7A2 expression levels (detected via antibodies)

  • Compare NO production in wild-type versus SLC7A2 knockout cells

• Protein Nitrosylation Analysis:

  • Examine protein nitrosylation patterns in relation to SLC7A2 expression

  • Focus on specific targets like dynamin-related protein-1 (Drp-1), which is involved in mitochondrial dynamics and known to be affected in Huntington's disease

• Live-Cell Imaging of Mitochondrial Dynamics:

  • Assess mitochondrial fragmentation in relation to SLC7A2 expression and arginine availability

  • This approach has been used to demonstrate the impact of altered arginine metabolism on mitochondrial function in HD cells

What technical challenges might researchers encounter when using SLC7A2 antibodies, and how can these be addressed?

Several technical challenges may arise when working with SLC7A2 antibodies:

• Molecular Weight Discrepancy:

  • Challenge: The observed molecular weight (~100 kDa) differs significantly from the calculated weight (~49.7 kDa)

  • Solution: This discrepancy is likely due to post-translational modifications or the glycosylated nature of membrane proteins. Researchers should be aware of this difference when interpreting western blot results.

• Isoform Detection:

  • Challenge: Different SLC7A2 isoforms have distinct functions but may be difficult to distinguish

  • Solution: Use isoform-specific antibodies when available, or combine with RT-PCR to confirm which isoforms are expressed in the system under study

• Membrane Protein Extraction:

  • Challenge: As a membrane transporter, SLC7A2 may be difficult to extract efficiently

  • Solution: Use specialized membrane protein extraction buffers containing appropriate detergents (e.g., CHAPS, NP-40)

• Fixation Sensitivity in IHC:

  • Challenge: Epitope accessibility may be affected by fixation methods

  • Solution: Compare different fixation protocols (paraformaldehyde, methanol) and consider antigen retrieval methods to optimize staining

• Background in Biotin-Rich Tissues:

  • Challenge: Endogenous biotin in tissues can interfere with biotin-conjugated antibody detection

  • Solution: Implement biotin blocking steps and include appropriate controls to distinguish specific from non-specific signal

How does SLC7A2 expression and function differ in Huntington's disease models compared to normal conditions?

Research has revealed significant alterations in SLC7A2 expression and function in Huntington's disease models:

• Expression Pattern Changes:

  • SLC7A2 is selectively upregulated in HD cellular models and patients

  • This upregulation was identified as one of the most significant gene expression changes when normal Huntingtin was deleted in human neuroblastoma SH-SY5Y cells using CRISPR-Cas9

• Functional Consequences:

  • HD cells exhibit an overactive response to neuroinflammatory challenges

  • Abnormally high inducible nitric oxide synthase (iNOS) induction and NO production occurs in HD models

  • This leads to increased protein nitrosylation

  • These effects can be blocked by either depleting extracellular arginine or knocking out SLC7A2

• Cellular Localization:

  • SLC7A2 is highly expressed in astrocytes and vascular and leptomeningeal cells in the brain

  • This expression pattern suggests cell-type specific roles in neuroinflammation

• Potential Therapeutic Implications:

  • Targeting SLC7A2 or modulating arginine availability may represent potential therapeutic approaches for HD

  • Preliminary exploratory studies have investigated the effect of arginine supplements on HD progression using the Enroll-HD periodic human patient dataset

What methods can be used to study the interaction between SLC7A2 and inflammatory pathways in neurological disorders?

To investigate SLC7A2's role in neuroinflammation, researchers can employ these methodological approaches:

• RNA and Protein Expression Analysis:

  • RNA sequencing (RNA-seq) and quantitative RT-PCR to assess SLC7A2 mRNA levels

  • Western blotting and immunohistochemistry using specific antibodies to quantify and localize protein expression

  • Data mining of publicly available RNA-seq datasets from human patients to correlate expression with disease severity

• Arginine Metabolism and Nitrosative Stress Assessment:

  • Biochemical assays to measure arginine uptake and metabolism

  • Analysis of nitric oxide production and protein nitrosylation in response to inflammatory stimuli

  • Evaluation of these parameters in cell lines and primary astrocytes to understand tissue-specific responses

• Genetic Manipulation Approaches:

  • CRISPR-Cas9 system for knocking out SLC7A2 in cellular models

  • siRNA or shRNA for transient knockdown studies

  • Overexpression systems to assess gain-of-function effects

  • These genetic tools allow direct assessment of SLC7A2's causal role in neuroinflammatory responses

• Live-Cell Imaging Techniques:

  • Visualization of mitochondrial dynamics in relation to SLC7A2 expression and function

  • Assessment of cellular stress responses and protein localization changes

  • Real-time monitoring of inflammatory marker expression

What are the key considerations when designing experiments to study SLC7A2 in macrophage activation and immune function?

When investigating SLC7A2's role in immune function, researchers should consider:

• Macrophage Polarization States:

  • SLC7A2 may play different roles in classical (M1) versus alternative (M2) activation of macrophages

  • Experimental design should include appropriate stimuli to induce different activation states:

    • M1 polarization: LPS, IFN-γ

    • M2 polarization: IL-4, IL-13

• Arginine Metabolism Pathways:

  • In macrophages, arginine can be metabolized through two major competing pathways:

    • iNOS pathway: Producing NO and citrulline (associated with M1 polarization)

    • Arginase pathway: Producing ornithine and urea (associated with M2 polarization)

  • Experiments should assess both pathways to fully understand SLC7A2's impact

• Cell Type Considerations:

  • Primary macrophages versus cell lines (differences in SLC7A2 expression and function)

  • Tissue-specific macrophage populations (microglia versus peripheral macrophages)

  • Species differences (human versus mouse versus rat) in SLC7A2 function and regulation

• Temporal Dynamics:

  • SLC7A2 expression and activity may change over the course of inflammatory responses

  • Time-course experiments are essential to capture these dynamics

  • Both acute and chronic inflammatory models should be considered

By addressing these considerations, researchers can design robust experiments to elucidate SLC7A2's specific contributions to immune function and inflammatory processes.

How might advances in antibody technology enhance the study of SLC7A2 in complex biological systems?

Emerging antibody technologies offer promising opportunities for advancing SLC7A2 research:

• Single-Cell Analysis Applications:

  • Combining SLC7A2 antibodies with single-cell technologies could reveal cell-specific expression patterns and heterogeneity

  • Mass cytometry (CyTOF) with metal-conjugated antibodies allows simultaneous detection of multiple markers alongside SLC7A2

  • These approaches would be particularly valuable for understanding SLC7A2's role in specific cell populations within heterogeneous tissues like brain or immune organs

• Super-Resolution Microscopy:

  • New imaging techniques combined with fluorophore-conjugated SLC7A2 antibodies could reveal subcellular localization with unprecedented precision

  • This would help clarify SLC7A2's spatial relationship with other membrane proteins and intracellular signaling components

• Antibody Engineering Approaches:

  • Development of isoform-specific antibodies with enhanced specificity

  • Creation of conformation-specific antibodies that distinguish between active and inactive transporter states

  • Bispecific antibodies to simultaneously target SLC7A2 and interacting proteins

• In vivo Imaging Applications:

  • Near-infrared fluorophore-conjugated antibodies for deeper tissue penetration

  • Development of smaller antibody fragments (Fab, nanobodies) with improved tissue access for in vivo studies

What are emerging research questions regarding SLC7A2's role in neuroinflammation and neurodegenerative diseases?

Current findings have opened several promising avenues for future investigation:

• Cell-Type Specific Functions:

  • How does SLC7A2 function differ between astrocytes, microglia, neurons, and vascular cells?

  • What are the consequences of cell-specific SLC7A2 knockout on neuroinflammation?

  • How does SLC7A2 expression in different cell types change during disease progression?

• Therapeutic Targeting Potential:

  • Could selective inhibition of SLC7A2 ameliorate neuroinflammation in Huntington's disease?

  • What are the systemic consequences of SLC7A2 modulation given its role in multiple tissues?

  • How do existing treatments for neurodegenerative diseases affect SLC7A2 expression and function?

• Translational Research Questions:

  • Can SLC7A2 expression serve as a biomarker for neuroinflammation in neurodegenerative diseases?

  • Do SLC7A2 polymorphisms correlate with disease susceptibility or progression?

  • What is the relationship between peripheral and central SLC7A2 expression in systemic inflammatory conditions?

• Mechanistic Investigations:

  • How does SLC7A2 upregulation specifically contribute to mitochondrial dysfunction in neurodegenerative diseases?

  • What transcriptional regulators control SLC7A2 expression in different cell types during inflammation?

  • How does SLC7A2 interact with other amino acid transporters to regulate cellular metabolism during stress?