Anti-CD133 Antibody, clone 13A4, Alexa Fluor™ 488 conjugated

Description

• General description: Prominin is a novel plasma membrane protein with an N-terminal extracellular domain, five transmembrane segments flanking two short cytoplasmic loops and two large glycosylated extracellular domains, and a cytoplasmic C-terminal domain. DNA sequences from Caenorhabditis elegans predict the existence of a protein with the same features, suggesting that prominin is conserved between vertebrates and invertebrates. Prominin is found not only in the neuroepithelium but also in various other epithelia of the mouse embryo. In the adult mouse, prominin has been detected in the brain ependymal layer, and in kidney tubules. In these epithelia, prominin is specific to the apical surface, where it is selectively associated with microvilli and microvilli-related structures. Remarkably, upon expression in CHO cells, prominin is preferentially localized to plasma membrane protrusions such as filopodia, lamellipodia, and microspikes. These observations imply that prominin contains information to be targeted to, and/or retained in, plasma membrane protrusions rather than the planar cell surface. Moreover, results show that the mechanisms underlying targeting of membrane proteins to microvilli of epithelial cells and to plasma membrane protrusions of non-epithelial cells are highly related (Weigmann et al., 1997). Also see (http://www.ncbi.nlm.nih.gov/prow/guide/438375806_g.htm) for more information on CD133 and prominin expression.
• Specificity: Recognizes mouse Prominin-1, a pentaspan transmembrane (5-TM) domain glycoprotein. Immunoblotting of E12 mouse brain and adult mouse kidney using mAb 13A4 showed that prominin has a mean apparent molecular weight of 115 kDa; Deglycosylation of brain and kidney prominin with peptide N-glycosidase F (PNGase F) yielded a 94-kDa band indicating that prominin is N-glycosylated. The additional 88-kDa band seen upon deglycosylation of brain prominin represents a C-terminally truncated form (Weigmann et al., 1997; Maw et al., 2000; Roper I, 2000). Prominin-1 is expressed on primitive cells such as hematopoietic stem and progenitor cells, neural & endothelial stem cells, retina and retinoblastoma, as well as developing epithelium. To date, the function and ligand of Prominin-1 are unknown. This antibody can be used to isolate murine stem cells from brain and bone marrow.
• Immunogen: Telencephali of ten 12-day-old NMRI mouse embryos (E12, ~1 mg protein), which consist mostly of neuroepithelium, were homogenized in 1 mL PBS, mixed with crushed nitrocellulose filter (1 cm2 in 0.5 mL PBS) as adjuvant, and injected i.p. into a LouXSD rat (Weigmann et al., PNAS 1997).
• Application: Immunohistochemistry: 1-15 μg/mL. Suggested fixatives: 2-4% PFA, or methanol -20°C fixation. When fixing cells in culture, incubate sample in 3% PFA for 15-30 min at room temperature. Tissue: perfusion with 2-4% PFA, 1-4 hours postfixation is typical. Traditional formalin fixation is NOT recommended. Permeabilization method: 0.2% saponin or 0.1-0.3% Triton X-100 in PBS. Blocking Buffer: For cells in culture, 0.2% gelatin in PBS; for tissue section, 10% FCS in PBS. Dilution Buffer: For cells in culture, 0.2% gelatin in PBS; for tissue section, 0.2% saponin and 10% FCS in PBS. Incubation Times/Temperature: Overnight at 4°C or 1 hour at 37°C. Note: This antibody does not work with paraffin-embedded sections. EM immunohistochemistry: The subcellular localization of the 13A4 antigen in mouse E9-10 neuroepithelial cells and adult kidney proximal tubule cells was investigated by immunogold electron microscopy. Strong labeling was observed over the kidney brush border membrane, where 13A4 immunoreactivity appeared to be concentrated toward the tips of the microvilli. Remarkably, in neuroepithelial cells, whose apical plasma membrane contains fewer microvilli than the kidney brush border, 13A4 immunoreactivity was associated mostly, if not exclusively, with microvilli and plasma membrane protrusions, and was not detected in the planar areas of the apical plasma membrane. Because of this preferential localization, the 13A4 antigen was referred to as ′′prominin′′ (from the Latin word ′′prominere,′′ to stand out, to be prominent) (Weigmann et al., 1997). Western blotting: 1-5 μg/mL in 0.3% Tween in PBS. Sample preparation: Standard Laemmli (boiled in 2% SDS, 100mM DTT or 5% beta-mercaptoethanol, 60mM Tris-HCL pH 6.8). Preferred Gel percentage: 7.5%. Suggested Blocking Buffer: 3-5% Milk, 0.3% Tween in PBS. Incubation time: 1 hour at room temperature or overnight at 4°C. Recommended control extracts: Positive: Kidney membrane; Negative: liver membranes. Immunoprecipitation: 10-25 μg/mL. Suggested tissue/cell lysis buffer: RIPA bufferFinal reaction volume: 500-1000 μL. Final total protein concentration in reaction mix: 0.5-3 mg/mL. Incubation times: overnight at 4°C. Capture agent used: Protein G Sepharose™ or rabbit anti-rat antibody/protein A Sepharose. Expected sizes on immunoblots (in kDa): 115 kDa (mature form) or 105 kDa (precursor form). FACS Analysis: Suggested dilution/number of cells: 0.25-1 μg/ million cells. Fixation/Permeabilization used: BD FACS lysis Solution (1-1.5% formaldehyde) (BD and FACS are trademarks of Becton, Dickinson and Company) No permeabilization. Recommended controls: Hematopoietic stem cells. Optimal working dilutions must be determined by the end user.
• Physical form: Liquid in Phosphate buffer with 15 mg/mL BSA as a stabilizer and 0.1% sodium azide.
• Storage and Stability: Maintain refrigerated at 2–8°C protected from light in undiluted aliquots for up to 24 months.
• Analysis Note: ControlHuman embryonic stem cell lysate, Caco 2 (Human colonic carcinoma cell line) whole cell lysate
• Other Notes: Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.
• Legal Information: ALEXA FLUOR is a trademark of Life Technologies
• Disclaimer: Alexa Fluor™ is a registered trademark of Molecular Probes, Inc.Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.