RAET1E antibodies are affinity-purified reagents designed to bind specifically to RAET1E, a type I transmembrane glycoprotein involved in triggering NKG2D receptor-mediated cytotoxicity in natural killer (NK) cells and cytotoxic T-lymphocytes . These antibodies are essential for studying RAET1E’s role in:
Immune surveillance against tumor cells and virally infected cells .
Stress responses linked to cellular proliferation, wound healing, and atherosclerosis .
Pathological conditions such as cancer and viral infections .
RAET1E antibodies vary in clonality, host species, and validated applications. Key examples include:
RAET1E antibodies are utilized across multiple experimental workflows:
ab200662 detects a 30 kDa band in human fetal brain lysates and HeLa cells, confirming RAET1E’s presence in epithelial and neural tissues .
MAB6285 identifies a 40 kDa band in HT-29 colon adenocarcinoma cells, highlighting RAET1E’s role in cancer biology .
MAB6285 distinguishes ULBP-4/RAET1E expression in HepG2 hepatocellular carcinoma cells, enabling quantification of surface ligand levels .
ab200662 efficiently pulls down RAET1E from HeLa lysates, facilitating downstream analysis of protein-protein interactions .
MAB6285 detects ULBP-4/RAET1E in ovarian cancer tissues, localizing expression to epithelial cell membranes .
RAET1E expression is directly regulated by E2F transcription factors, which drive its transcription during cellular proliferation . Nuclear run-on assays revealed that proliferating cells exhibit elevated Raet1e transcription compared to serum-starved cells .
Raet1e is implicated in atherosclerosis susceptibility. Mouse studies identified a mutation in the Raet1e promoter linked to reduced expression and delayed atherosclerosis progression .
RAET1E activates NKG2D-bearing NK cells to target malignant cells. Antibodies like ab95202 (79/6/13/16) have been used to study RAET1E’s role in tumor immune evasion mechanisms .
Herpes simplex virus type 1 (HSV-1) induces RAET1E expression by inhibiting histone deacetylase 3 (HDAC3), which represses Raet1e under basal conditions .
Antibody | Applications | Species Reactivity | Key Validation |
---|---|---|---|
ab200662 | WB, IP | Human, Mouse, Rat | 30 kDa band in HeLa lysates |
MAB6285 | WB, Flow Cyt, IHC | Human | 40 kDa band in HT-29 cells |
ab95202 | IP, Flow Cyt, ELISA | Human | Cited in 5 publications |
Condition | RAET1E Role | Antibody Used | Mechanism |
---|---|---|---|
Cancer | Ligand for NK cell activation | ab95202, MAB6285 | NKG2D-mediated cytotoxicity |
Atherosclerosis | Endothelial cell stress marker | N/A (mouse models) | Promotes macrophage infiltration |
Viral Infection | Stress-induced immune response | ab200662, MAB6285 | HDAC3 inhibition enhances expression |
RAET1E (ULBP4) belongs to the ULBP/RAET1 family of cell surface proteins that function as ligands for NKG2D receptors. Unlike most family members which are GPI-anchored, RAET1E expresses a transmembrane form, making it structurally distinct . Human RAET1E mRNA encodes 263 amino acids including a 30 amino acid signal sequence, a 195 amino acid extracellular domain, a 23 amino acid transmembrane domain, and a 15 amino acid cytoplasmic sequence .
RAET1E serves as a functional ligand for NKG2D, triggering lymphocyte activation that results in cytokine secretion . Its significance lies in its differential expression pattern between normal and pathological tissues, being abnormally expressed in various cancer types, particularly colon cancer . This makes RAET1E a valuable target for understanding immune surveillance mechanisms in cancer progression and potential immunotherapeutic approaches.
Based on current research tools, RAET1E antibodies are available in multiple formats tailored to specific experimental needs:
When selecting an antibody, researchers should consider the specific experimental requirements, including application compatibility, species reactivity (primarily human for most commercial RAET1E antibodies), and the specific epitope recognition needed for their study.
Western blot optimization for RAET1E detection requires careful consideration of several technical parameters:
Sample preparation: For optimal results with RAET1E detection, use PVDF membrane and prepare lysates from appropriate cell lines such as HT-29 human colon adenocarcinoma cells, where RAET1E expression has been well-documented .
Antibody concentration: Based on published protocols, use a primary antibody concentration of approximately 1 μg/mL (e.g., Mouse Anti-Human ULBP-4/RAET1E Monoclonal Antibody) .
Detection conditions: Conduct experiments under reducing conditions using appropriate buffer systems (e.g., Immunoblot Buffer Group 1) .
Expected molecular weight: Look for a specific band for RAET1E at approximately 40 kDa, though variations between 40-50 kDa have been reported due to post-translational modifications .
Controls: Include appropriate positive controls such as HT-29 cell lysates and negative controls to validate antibody specificity.
If signal intensity is suboptimal, consider extending primary antibody incubation time (overnight at 4°C), optimizing blocking conditions, or exploring alternative antibody clones that may offer higher sensitivity for Western blot applications.
Flow cytometry using RAET1E antibodies requires specific optimization strategies:
Cell preparation: Fresh, viable single-cell suspensions yield optimal results. For RAET1E detection, HepG2 human hepatocellular carcinoma cell line has been validated as an appropriate positive control .
Antibody titration: Perform titration experiments to determine optimal antibody concentration for your specific cell type.
Appropriate controls: Always include isotype control antibodies (e.g., MAB0041) to establish specific binding and assess background fluorescence .
Secondary antibody selection: For unconjugated primary antibodies, select appropriate fluorophore-conjugated secondary antibodies (e.g., Allophycocyanin-conjugated Anti-Mouse IgG) .
Gating strategy: Establish proper gating based on forward/side scatter properties, followed by singlet discrimination and viability assessment before analyzing RAET1E expression.
Remember that RAET1E expression can be modulated by various factors including TNF-alpha (increases expression) and retinoic acid (decreases expression) , which may impact experimental outcomes depending on culture conditions.
Immunohistochemistry (IHC) with RAET1E antibodies requires specific optimization for clinical samples:
Tissue preparation: Use immersion-fixed paraffin-embedded sections. RAET1E has been successfully detected in human ovarian cancer tissue using this method .
Antibody concentration: A concentration of 15 μg/mL with overnight incubation at 4°C has proven effective for RAET1E detection in tissue sections .
Detection system: HRP-DAB Cell & Tissue Staining Kit provides good results for visualizing RAET1E expression, with hematoxylin counterstaining to provide cellular context .
Localization pattern: Expect specific staining localized to plasma membranes of epithelial cells when detecting RAET1E, as this is a membrane-bound protein .
Troubleshooting: If non-specific staining occurs, increase blocking time, optimize antibody dilution, or consider antigen retrieval method adjustments.
When analyzing clinical samples, researchers should note that RAET1E expression is typically low in normal tissues but may be elevated in certain cancers, making it important to include appropriate positive and negative control tissues for accurate interpretation.
Several methodological challenges exist when studying RAET1E in cancer contexts:
Variable expression levels: RAET1E expression can vary significantly between cancer types and even within the same tumor type, requiring careful sampling strategies and quantitative analysis methods .
Soluble vs. membrane-bound forms: A soluble 35 kDa form of RAET1E exists that may antagonize the transmembrane form, complicating interpretation of results . Researchers must distinguish between these forms using appropriate antibodies and experimental designs.
Splice variants: Multiple potential splice variants (220, 227, and 280 amino acids) exist as transmembrane proteins , potentially affecting antibody recognition depending on the epitope targeted.
Expression regulation: RAET1E expression is dynamically regulated by factors such as TNF-alpha and retinoic acid , requiring careful consideration of experimental conditions that might artificially alter expression patterns.
Tissue-specific considerations: While RAET1E has been detected in colon cancer and ovarian cancer samples , expression patterns may differ in other malignancies, necessitating validation in each specific cancer type under investigation.
RAET1E expression is subject to complex transcriptional regulation that researchers should consider when designing experiments:
Promoter structure: The RAET1E promoter contains specific response elements that regulate its expression. Research has identified critical regions within the first 95-85 bp upstream of the transcription start site that are essential for transcriptional activation .
Transcription factor binding: Specificity factor (Sp) transcription factor family binding sites have been identified in the RAET1E promoter. Specifically, Sp3 has been shown to constitutively occupy the RAET1E promoter .
Experimental approaches: For studying RAET1E transcriptional regulation, researchers can employ techniques such as:
Functional validation: Dominant negative approaches targeting Sp transcription factors have demonstrated their importance in RAET1E expression regulation, suggesting a mechanistic approach for experimental manipulation .
When investigating RAET1E expression patterns in experimental models, researchers should consider these transcriptional regulatory mechanisms, particularly when interpreting seemingly contradictory results between RNA and protein levels.
Several approaches can be employed to investigate the impact of genetic variations on RAET1E expression and function:
Promoter analysis: Using dual-luciferase reporter assays to compare the activity of different RAET1E promoter variants. This approach has successfully demonstrated functional differences in promoter activity related to sequence variations in the transcription initiation region .
Site-directed mutagenesis: Introducing specific mutations into the RAET1E promoter to evaluate their functional impact on gene expression. This technique can isolate the effects of individual SNPs (Single Nucleotide Polymorphisms) .
Transgenic models: Development of RAET1E transgenic mice has provided valuable insights into the role of RAET1E as a modifier gene in conditions such as atherosclerosis .
Cell line models: Different cell lines (e.g., C57SV002, BalbcSV006, NIH3T3) can be used to assess the impact of RAET1E variants in different cellular contexts .
These methodological approaches have revealed that genetic variations in RAET1E can have significant functional consequences, including altered expression patterns that may influence disease susceptibility and progression.
Recent research has identified RAET1E as a novel atherosclerosis modifier gene , opening new avenues for investigation using RAET1E antibodies:
Expression analysis in vascular tissue: Use immunohistochemistry with RAET1E antibodies to assess expression patterns in atherosclerotic lesions compared to normal vessels. The protocol using 15 μg/mL antibody concentration with overnight incubation at 4°C has proven effective for tissue staining .
Cellular models: Apply flow cytometry with RAET1E antibodies to analyze expression in relevant cell types (endothelial cells, smooth muscle cells, macrophages) under atherogenic conditions.
Genetic association studies: Combine genetic data on RAET1E variants with protein expression analysis using specific antibodies to create genotype-phenotype correlations in atherosclerosis.
Mechanistic investigations: Utilize RAET1E antibodies in co-immunoprecipitation experiments to identify interaction partners that may explain its role in atherosclerosis progression.
Functional blockade: Consider using neutralizing antibodies against RAET1E in experimental models to assess the functional importance of this protein in atherosclerotic processes.
This emerging area of research connects immune recognition molecules like RAET1E with cardiovascular disease, requiring interdisciplinary approaches that combine immunological and cardiovascular research methodologies.
Rigorous validation of RAET1E antibodies is critical for reliable experimental results:
Positive and negative controls: Use cell lines with confirmed RAET1E expression (positive controls such as HT-29 colon adenocarcinoma cells or HepG2 cells ) and those lacking expression (negative controls) to validate antibody specificity.
Multiple detection methods: Confirm results using at least two independent methods (e.g., Western blot and flow cytometry) to increase confidence in antibody specificity.
Isotype controls: Always include appropriate isotype controls in flow cytometry experiments to differentiate specific binding from background or Fc receptor binding .
Blocking peptides: Use recombinant RAET1E protein or the immunizing peptide (e.g., recombinant His-tagged protein to human extra-cellular domain of RAET1E ) as a competitive inhibitor to confirm binding specificity.
Knockdown/knockout validation: When possible, validate antibody specificity using RAET1E knockdown or knockout models to demonstrate reduced or absent signal.
Cross-reactivity assessment: Evaluate potential cross-reactivity with other ULBP/RAET1 family members, particularly those sharing sequence identity (RAET1E shares 34-41% amino acid sequence identity with family members ).
Thorough validation not only ensures experimental rigor but also enhances reproducibility and reliability of RAET1E-focused research findings.