SLC38A9 Antibody, Biotin conjugated

What is SLC38A9 and what is its biological significance?

SLC38A9 (Solute Carrier Family 38 Member 9) is a lysosomal amino acid transporter that functions as a critical component of the amino acid-sensing machinery controlling mTORC1 (mammalian Target of Rapamycin Complex 1) signaling. The protein is also known as Sodium-coupled neutral amino acid transporter 9 and has been identified as an integral component of the Ragulator/RAG GTPase complex at the lysosomal membrane. SLC38A9 acts primarily as an amino acid sensor rather than a high-capacity transporter, with particular sensitivity to arginine levels in lysosomes .

The significance of SLC38A9 lies in its role as a physical and functional component of the lysosomal amino acid sensing complex that regulates cell growth in response to nutrient availability. Recent research has also implicated SLC38A9 in viral pathogenesis, as it may regulate SARS-CoV-2 viral entry into host cells .

What applications has SLC38A9 Antibody, Biotin conjugated been validated for?

When designing experiments with SLC38A9 Antibody, Biotin conjugated, researchers should perform preliminary validation studies for applications beyond ELISA .

What are the optimal storage and handling conditions for maintaining antibody activity?

For optimal preservation of SLC38A9 Antibody, Biotin conjugated activity, the following storage and handling conditions are recommended:

• Store the antibody at -20°C or -80°C to maintain stability .

• Upon delivery, aliquot the antibody to minimize freeze-thaw cycles, as repeated freezing and thawing can degrade antibody performance .

• The antibody is typically provided in storage buffer containing 50% Glycerol and 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative .

• For long-term storage beyond one year, -80°C is preferable over -20°C.

Proper handling of the antibody is critical for experimental success, as improper storage can lead to reduced sensitivity and specificity in downstream applications .

How should I optimize immunoprecipitation protocols when studying SLC38A9 interactions with the Ragulator/RAG GTPase complex?

When designing immunoprecipitation experiments to study SLC38A9 interactions with the Ragulator/RAG GTPase complex, several critical considerations must be addressed:

• Sample preparation challenges: SLC38A9 has been observed to form insoluble aggregates upon boiling, making it difficult to analyze using standard SDS-PAGE techniques. Use a gel-free approach with liquid chromatography tandem mass spectrometry (LC-MS/MS) for optimal results .

• Selective enrichment strategy: When co-immunoprecipitating SLC38A9 with components of the Ragulator/RAG GTPase complex, both approaches are valuable:

  • Pull-down of SLC38A9 to detect LAMTOR1, RAGA, and other complex components

  • Pull-down of RAGA or LAMTOR proteins to confirm the presence of SLC38A9

• Validation controls: Include antibodies against other SLC38 family members (SLC38A1, SLC38A2, SLC38A7) and SLC36 family members (SLC36A1/PAT1, SLC36A4/PAT4) as negative controls to confirm specificity of interactions .

• Detection sensitivity: The sequence coverage of SLC38A9 in mass spectrometry analyses is typically lower than other complex components due to inefficient proteolytic cleavage of transmembrane regions. Plan accordingly when analyzing results .

What methodological approaches are most effective for studying SLC38A9's role in amino acid sensing?

To effectively investigate SLC38A9's function in amino acid sensing, researchers should consider these methodological approaches:

• Domain-specific analysis: The N-terminal cytoplasmic tail (amino acids 1-112, specifically residues 31-112) is sufficient and required for binding to the Ragulator/RAG GTPases complex. Design experiments targeting this region when studying protein interactions .

• Mutational studies: Four conserved motifs have been identified in the N-terminal region. Mutation of any of the first three motifs completely abolishes binding to the complex, while disruption of the fourth has no effect. These can serve as useful tools for structure-function studies .

• Conformational state analysis: The interaction of SLC38A9 with RAG GTPases is dramatically influenced by their nucleotide-binding state. RAGA T21N and RAGB T54N mutants show increased SLC38A9 recruitment, while RAGC S75N abolishes binding. Consider this when designing experiments to capture dynamic interactions .

• Subcellular localization confirmation: Always validate lysosomal localization in your experimental system using co-localization studies with markers such as LAMP1, CD63, and LBPA to ensure proper targeting of SLC38A9 .

How can I address the technical challenges of SLC38A9 protein aggregation during sample preparation?

SLC38A9 is prone to forming insoluble aggregates that fail to enter SDS-polyacrylamide gels upon boiling, creating significant challenges for standard protein analysis techniques . To overcome these issues:

• Alternative lysis methods:

  • Use non-denaturing lysis buffers containing mild detergents like CHAPS or digitonin

  • Consider room temperature solubilization rather than boiling samples

  • If denaturing conditions are necessary, use 8M urea-based buffers instead of SDS/heat

• Gel-free proteomics approach:

  • Implement one-dimensional gel-free liquid chromatography tandem mass spectrometry (LC-MS/MS)

  • This approach has proven critical for successfully analyzing SLC38A9 complexes

• Sample handling optimization:

  • Process samples quickly to minimize aggregation time

  • Consider adding low concentrations of reducing agents to prevent disulfide bond formation

  • Pre-clear lysates by high-speed centrifugation to remove pre-formed aggregates

• Western blotting modifications:

  • Use gradient gels with larger pore sizes to facilitate entry of any partially solubilized protein

  • Transfer to PVDF membranes at lower voltage for extended periods

  • Consider detecting SLC38A9 fragments rather than the full-length protein

What explains the discrepancy between the calculated and observed molecular weights of SLC38A9?

SLC38A9 exhibits interesting differences between its theoretical and observed molecular weights that researchers should be aware of:

Several factors may contribute to this discrepancy:

• Post-translational modifications: The protein may undergo processing that affects its migration pattern in gels.

• Alternative splicing: Multiple isoforms (64, 53, and 57 kDa) have been reported, likely resulting from alternative splicing events .

• Protein folding and hydrophobicity: The transmembrane regions of SLC38A9 may bind more SDS and affect migration.

• Proteolytic processing: Some evidence suggests the protein may be cleaved during cellular trafficking or function.

Researchers should validate which isoform they are detecting in their experimental system and consider the implications for functional studies .

How can I optimize immunofluorescence protocols for studying SLC38A9 localization?

Optimizing immunofluorescence protocols for SLC38A9 localization studies requires careful attention to several key factors:

• Fixation method selection:

  • Paraformaldehyde (4%) is generally preferred for maintaining membrane protein structure

  • Avoid methanol fixation as it can disrupt membrane protein epitopes

• Permeabilization optimization:

  • Use mild detergents (0.1% Triton X-100 or 0.1% saponin)

  • For selective permeabilization of plasma membrane while preserving lysosomal membranes, consider digitonin titration

• Co-localization markers:

  • Include established lysosomal markers: LAMP1, CD63, LBPA

  • Include negative controls: EEA1 (early endosomes), Giantin (Golgi)

  • These will confirm proper localization to late endosomes/lysosomes

• Antibody dilution and incubation:

  • Start with recommended dilution range of 1:50-1:500

  • Perform a titration experiment to determine optimal concentration

  • Extend primary antibody incubation to overnight at 4°C for better penetration

For confirming SLC38A9 lysosomal localization, extensive colocalization with late endosome/lysosome markers has been observed, but not with early endosome or Golgi markers, supporting its function as a component of the lysosomal amino acid sensing machinery .

What experimental approaches are most effective for studying SLC38A9's role in mTORC1 activation?

To effectively study SLC38A9's role in mTORC1 activation, researchers should consider these strategic approaches:

• Amino acid starvation and refeeding experiments:

  • SLC38A9 functions in the amino acid sensing machinery controlling mTORC1

  • Design protocols with complete amino acid starvation followed by selective refeeding

  • Monitor phosphorylation of mTORC1 substrates (e.g., S6K, 4EBP1) under various conditions

• Mutational analysis of key domains:

  • The N-terminal domain (amino acids 31-112) is critical for interaction with the Ragulator/RAG GTPase complex

  • Create mutations in the three identified conserved motifs that abolish binding

  • These mutants retain lysosomal localization but disrupt complex formation, providing excellent tools for functional studies

• Dynamic complex assembly assessment:

  • The interaction of SLC38A9 with RAG GTPases is dramatically influenced by their nucleotide-binding state

  • RAGA T21N and RAGB T54N (GDP-bound) mutants show increased SLC38A9 recruitment

  • RAGC S75N abolishes binding, while GTP-bound RAGA Q66L/B Q99L mutants show reduced binding

  • These findings suggest SLC38A9 interacts specifically with certain conformational states of the complex

• Lysosomal vs. plasma membrane amino acid sensing:

  • SLC38A9 participates in mTORC1 activation at lysosomes rather than at the plasma membrane

  • Unlike transporters that import extracellular amino acids, SLC38A9 levels are not typically induced upon amino acid starvation

  • Design experiments that distinguish between these compartments to accurately assess function

How can SLC38A9 Antibody be used to investigate potential roles in viral pathogenesis?

Recent research has implicated SLC38A9 in SARS-CoV-2 viral entry, opening a new avenue for investigation . To explore this relationship using SLC38A9 antibodies:

• Viral entry inhibition studies:

  • Use antibody-mediated blocking of SLC38A9 in cell culture models

  • Assess changes in viral entry efficiency through quantitative PCR or immunofluorescence

  • Compare results with established entry inhibitors targeting ACE2 or TMPRSS2

• Mechanism investigation through co-localization:

  • Examine co-localization of SLC38A9 with viral components during entry

  • Use dual immunofluorescence with SLC38A9 antibody and antibodies against viral proteins

  • Perform time-course experiments to track the dynamics of interaction

• Proteolytic processing analysis:

  • SARS-CoV-2 entry depends on spike protein cleavage into S1 and S2 by furin

  • Investigate whether SLC38A9 influences this process directly or indirectly

  • Use Western blotting to detect changes in spike protein processing in the presence of SLC38A9 blocking antibodies

• Structure-function relationship:

  • Determine which domains of SLC38A9 are involved in viral entry

  • Create domain-specific blocking antibodies or use epitope mapping

  • This information could guide development of therapeutic interventions

The investigation of SLC38A9's role in viral pathogenesis represents an emerging area of research that may reveal new insights into both viral mechanisms and the broader functions of this protein beyond amino acid sensing.

What are the key considerations when interpreting SLC38A9 knockout or knockdown experiments?

When designing and interpreting SLC38A9 knockout or knockdown experiments, researchers should consider several important factors:

Understanding these considerations will ensure more accurate interpretation of experimental results and avoid common pitfalls in the analysis of this complex signaling pathway.

How do different commercially available SLC38A9 antibodies compare for specific research applications?

When selecting an SLC38A9 antibody for specific research applications, consider these comparative features:

What are the critical differences between studying endogenous versus overexpressed SLC38A9?

Understanding the differences between studying endogenous versus overexpressed SLC38A9 is essential for accurate interpretation of results:

• Protein localization considerations:

  • Endogenous SLC38A9: Primarily localizes to lysosomes with native trafficking patterns

  • Overexpressed SLC38A9: May show partial mislocalization or saturation of trafficking machinery

  • Recommendation: Always verify lysosomal localization of overexpressed constructs using markers such as LAMP1, CD63, and LBPA

• Complex formation dynamics:

  • Endogenous SLC38A9: Forms physiologically relevant complexes with native stoichiometry

  • Overexpressed SLC38A9: May alter complex stoichiometry or form non-physiological interactions

  • Evidence: Immunoprecipitation of SLC38A9 recruits endogenous RAGA and LAMTOR1, confirming complex membership at endogenous levels in multiple cell lines (HeLa, K562, NIH/3T3, RAW 264.7)

• Functional impact assessment:

  • Endogenous SLC38A9: Reflects physiological regulation of mTORC1 signaling

  • Overexpressed SLC38A9: May bypass normal regulatory mechanisms or create dominant-negative effects

  • Validation approach: Compare results between endogenous studies and carefully titrated expression systems

• Detection sensitivity trade-offs:

  • Endogenous SLC38A9: More challenging to detect but provides physiologically relevant data

  • Overexpressed SLC38A9: Easier detection but potential artifacts

  • Solution: Use tagged inducible expression systems with titratable expression levels

For optimal experimental design, researchers should consider using inducible expression systems that allow controlled expression levels or CRISPR-based endogenous tagging approaches to maintain physiological regulation while enhancing detection.