PROMININ-1 (CD133); ALEXA488; 100UG

Stock Code: 3572018
Manufacturer Part No: MAB4310X

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Descriptions

Analysis Note

ControlHuman embryonic stem cell lysate, Caco 2 (Human colonic carcinoma cell line) whole cell lysate

Application

Anti-CD133 Antibody, clone 13A4, Alexa Fluor 488 conjugated is an antibody against CD133 for use in FC.

Research CategoryStem Cell Research

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.

Research Sub CategoryNeural Stem CellsHematopoietic Stem Cells

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