ormdl1 Antibody

What is ORMDL1 and why is it significant in research?

ORMDL1 belongs to the ORM1-like family of proteins localized to the endoplasmic reticulum membrane. It serves as a negative regulator of sphingolipid synthesis by modulating the activity of serine palmitoyltransferase (SPT), the rate-limiting enzyme in sphingolipid biosynthesis . ORMDL1 plays essential roles in homeostatic regulation of sphingolipid de novo biosynthesis in response to ceramide levels . Research significance includes:

• Connection to gamma-secretase complex maturation

• Involvement in Presenilin expression (implicated in Alzheimer's disease)

• High conservation across species, suggesting evolutionary importance

• Regulatory functions in lipid metabolism

What applications are validated for commercial ORMDL1 antibodies?

Commercial ORMDL1 antibodies have been validated for multiple applications with specific recommended dilutions:

Researchers should validate each antibody in their specific experimental system as performance may vary between tissues and conditions .

How should researchers select the appropriate ORMDL1 antibody?

Selection criteria should include:

• Immunogen location: Choose antibodies targeting different epitopes if comparing with other ORMDL family members. For example, some antibodies target amino acids 48-100 of ORMDL1 , while others target peptides within amino acids 50-100 .

• Species reactivity: Human ORMDL1 shares 99% identity with mouse ORMDL1, allowing cross-reactivity in many cases .

• Validation data: Review immunoblot and immunohistochemistry images provided by vendors to confirm specific binding at the predicted molecular weight (17 kDa) .

• Application-specific validation: Some antibodies perform better in certain applications—review application-specific data rather than assuming uniform performance across methods .

How can researchers optimize detection specificity when studying ORMDL family members?

The high sequence homology between ORMDL family members (80-84% identity between human ORMDL1, ORMDL2, and ORMDL3) creates challenges for specific detection. Optimization strategies include:

• Epitope selection: Use antibodies targeting regions with lower conservation between family members

• Validation in knockout systems: Test antibody specificity in ORMDL1 knockout models to confirm absence of signal

• Cross-reactivity testing: Preabsorb antibodies with recombinant ORMDL2 and ORMDL3 to reduce cross-reactivity

• Complementary detection methods: Combine antibody-based detection with mRNA analysis to confirm specificity

• Western blot optimization: Use higher percentage gels (15-18%) to better separate the similarly-sized ORMDL proteins

In competitive bone marrow transplantation assays, researchers have confirmed ORMDL1 antibody specificity by demonstrating differential phenotypes between wild-type and Ormdl1-/-/Ormdl3-/- cells .

What methodological approaches can detect changes in ORMDL1 subcellular localization?

ORMDL1 primarily localizes to the endoplasmic reticulum but can redistribute under specific stimuli. For example, cholesterol loading induces ORMDL1 redistribution from the ER to cytoplasmic p62-positive autophagosomes . Effective detection methods include:

• Subcellular fractionation: Separate ER, cytosolic, and autophagosomal fractions before immunoblotting

• Confocal microscopy: Use dual immunofluorescence with ER markers (e.g., calnexin) and ORMDL1 antibodies

• Live-cell imaging: When possible, use fluorescently tagged ORMDL1 to track dynamic changes

• Immunoelectron microscopy: For highest resolution localization studies

• Proximity ligation assays: To confirm protein-protein interactions between ORMDL1 and potential binding partners

Research has shown that immunoprecipitation with ORMDL1 antibodies can detect increased association with autophagy proteins like p62 under specific conditions such as cholesterol loading .

How can ORMDL1 antibodies be utilized to investigate sphingolipid metabolism?

ORMDL1 regulates sphingolipid synthesis by inhibiting serine palmitoyltransferase. Research approaches include:

• Co-immunoprecipitation studies: Using ORMDL1 antibodies to pull down SPT complexes (SPTLC1/SPTLC2) and analyze interactions under various conditions

• Quantitative immunoblotting: Correlate ORMDL1 protein levels with sphingolipid measurements by mass spectrometry

• Immunofluorescence co-localization: Visualize ORMDL1 with SPT complex components

• Cholesterol loading experiments: Monitor changes in ORMDL1 levels and SPT activity simultaneously

• Pharmacological interventions: Track ORMDL1 expression after treatment with compounds that alter sphingolipid metabolism

Research has demonstrated that simultaneous deletion of ORMDL1 and ORMDL3 disrupts sphingolipid homeostasis, which can be monitored by both antibody-based approaches and mass spectrometry .

What controls should be implemented when using ORMDL1 antibodies?

Robust experimental design requires appropriate controls:

• Positive controls: Cell lines with confirmed ORMDL1 expression (e.g., SK-N-SH cells)

• Negative controls:

  • Isotype controls (rabbit IgG) to assess non-specific binding

  • ORMDL1 knockout or knockdown samples when available

  • Blocking peptide competition to confirm specificity

• Loading controls: Use ER resident proteins for normalization when studying ORMDL1

• Cross-reactivity assessment: Test antibody against recombinant ORMDL2 and ORMDL3 proteins

How should researchers interpret conflicting ORMDL1 antibody results?

Inconsistencies between experiments may result from:

• Epitope accessibility: The membrane topology of ORMDL1 (with both N and C termini on the cytosolic face of ER) may affect epitope accessibility in different sample preparations

• Post-translational modifications: Unknown modifications may alter antibody recognition

• Family cross-reactivity: Some antibodies may cross-react with ORMDL2/3 despite vendor claims

• Degradation conditions: ORMDL1 can be degraded through autophagy under specific conditions (e.g., cholesterol loading)

Recommended approaches:

• Use multiple antibodies targeting different epitopes

• Employ complementary techniques (e.g., mass spectrometry) to verify results

• Document experimental conditions precisely, including detergent concentrations for membrane protein extraction

What factors affect ORMDL1 stability in experimental samples?

ORMDL1 protein stability is affected by several factors that researchers should consider:

• Free cholesterol levels: Excess cholesterol increases ORMDL1 turnover through autophagy

• Preservatives: Standard preservatives (0.02-0.03% sodium azide or Proclin 300) in antibody solutions don't affect ORMDL1 detection

• Storage buffers: Glycerol-containing buffers (typically 50%) help maintain antibody performance for ORMDL1 detection

• Sample preparation: ORMDL1's membrane localization requires appropriate detergent extraction methods

• Autophagy inhibitors: Chloroquine or ATG7 knockdown prevents ORMDL1 degradation in cholesterol-loaded cells

How can ORMDL1 antibodies contribute to Alzheimer's disease research?

ORMDL1 has been implicated in Presenilin (PSI) expression, which is involved in Alzheimer's disease (AD) . Research approaches include:

• Expression analysis: Compare ORMDL1 levels in AD versus control brain tissues

• Co-localization studies: Examine spatial relationships between ORMDL1 and Presenilin using dual immunofluorescence

• Gamma-secretase complex analysis: Use ORMDL1 antibodies for immunoprecipitation to study interactions with gamma-secretase components

• Knockout models: Compare AD pathology markers in wild-type versus ORMDL1-deficient models

The finding that ORMDL1 expression is down-regulated in PSI mutations suggests its potential as a therapeutic target for Alzheimer's disease .

What approaches can differentiate the roles of ORMDL1 versus other family members in immune cell development?

Recent research has shown that simultaneous deletion of ORMDL1 and ORMDL3 disrupts B cell development, highlighting their importance in immune function :

• Competitive bone marrow transplantation: Using antibodies to track donor cell fate in recipient animals

• Flow cytometry: Combining surface markers with intracellular ORMDL1 staining to evaluate expression in immune subsets

• Single-cell analysis: Correlating ORMDL1 expression with developmental states

• Sphingolipid profiling: Measuring how ORMDL1 deficiency alters sphingolipid compositions in immune cells

• Homing assays: Tracking labeled cells to determine if ORMDL1 affects immune cell migration patterns

Research has demonstrated that ORMDL1/ORMDL3 double knockout impacts B cell development in a manner dependent on lymphocyte intrinsic factors rather than homing defects .

How might ORMDL1 antibodies be utilized in sphingolipid-related disease research beyond Alzheimer's?

ORMDL proteins have been implicated in various conditions including asthma :

• Tissue-specific expression analysis: Comparing ORMDL1 levels across affected versus unaffected tissues

• Genetic correlation studies: Combining antibody-based protein quantification with genetic association data

• Therapeutic intervention monitoring: Tracking ORMDL1 expression changes following treatments that modify sphingolipid metabolism

• Biomarker development: Evaluating ORMDL1 as a potential biomarker for disease progression or treatment response

The connection between ORMDL family proteins and childhood asthma suggests broader implications for ORMDL1 in respiratory and immune-related conditions .

What methodological innovations might improve ORMDL1 detection sensitivity?

Current and emerging approaches to enhance detection include:

• Proximity-based methods: Using split reporter systems or FRET-based approaches to study ORMDL1 interactions with greater sensitivity

• Mass spectrometry immunoprecipitation: Combining immunoprecipitation with mass spectrometry for precise identification

• Super-resolution microscopy: Examining ORMDL1 distribution within the ER at nanoscale resolution

• Quantitative approaches: Developing calibrated assays for absolute quantification of ORMDL1 protein levels

• Single-molecule tracking: Monitoring individual ORMDL1 molecules to study dynamics in live cells

The continued development of these methods will provide researchers with more sensitive and specific tools for studying ORMDL1 biology.