Anti-CD133, clone 13A4

Anti-CD133, clone 13A4

Chemicon (Millipore)

Recognizes mouse Prominin-1 (sometimes also referred to as CD133 and, in the case of the human orthologue, as AC133), a 115-120 kDa pentaspan transmembrane (5-TM) domain glycoprotein. 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.

The 13A4 antibody does not cross react with rat, human, chicken, or Drosophila antigen. The original paper is located at: http://www.pnas.org/cgi/reprint/94/23/12425.pdf

115 kDa

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}

13A4

IgG1κ

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, A et al.1997}. Also see {http://www.ncbi.nlm.nih.gov/prow/guide/438375806_g.htm} for more information on CD133 and prominin expression.

Mouse

• Chicken
• Drosophila
• Rat

Immunohistochemistry: Recommended antibody dilution: 1-15 μg/ml
(Does not work in paraffin embedded specimens)

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

Recommended control tissue: Positive: Kidney proximal tubules or epididymis; Negative: liver

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, we propose to call the 13A4 antigen 'prominin' (from the Latin word 'prominere,' to stand out, to be prominent). {http://www.pnas.org/cgi/reprint/94/23/12425.pdf}.

Western blotting: Recommended primary antibody dilution: 1-5 μg/ml in 0.3% Tween in PBS. Sample preparation: Standard Laemmli (SDS, reducing agents, boiled {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.

Prominin is a membrane-associated 115-kDa 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 http://www.pnas.org/cgi/reprint/94/23/12425.pdf} also see {Maw, 2000 http://hmg.oupjournals.org/cgi/content/full/9/1/27 or http://www.nature.com/ncb/journal/v2/n9/extref/ncb0900_582-s1.pdf

Recommended control extracts: Positive: Kidney membrane; Negative: liver membranes.

Immunoprecipitation: Suggested antibody dilution/amount: 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 FACSTM 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 end user.

Immunohistochemistry (Paraffin)

Positive Control: Kidney tissue or membranes
Negative Control: Liver tissue or membranes

Protein A purfied

Protein A Purified mouse immunoglobulin in 20 mM sodium phosphate, 250 mM NaCl, pH. 7.6, with 0.1% sodium azide as a preservative.

Maintain for 1 year at 2–8°C from date of shipment. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.

O43490

NM_006017.1

• PROM1
• CD133
• AC133
• MSTP061
• hProminin
• PROML1

Prominin-1; AC133 (human)

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.

• Flow Cytometry
• Immunohistochemistry
• Immunoprecipitation
• Western Blotting

The CD133 gene codes for a pentaspan transmembrane glycoprotein. The CD133 antigen appears to belong to a new molecular family of 5-TM proteins, as the characterization of the CD133 antigen and prominin in the mouse were the first descriptions of a 5-TM glycoprotein structure. This "family" includes members from several different species (which may be homologs) including human, mouse, rat, fly, and worm. The 5-TM structure includes an extracellular N-terminus, two short intracellular loops, two large extracellular loops and an intracellular C-terminus CD133 was initially shown to be expressed on primitive hematopoietic stem and progenitor cells and retinoblastoma. CD133 has since been shown to be expressed on hemangioblasts, and neural stem cells as well as on developing epithelium. Expression patterns for CD133 generally mimic those of the murine prominin molecule, although CD133 antigen has not yet been demonstrated on adult epithelial tissue. The CD133 positive fraction of human bone marrow, cord blood and peripheral blood have been shown to efficiently engraft in xenotransplantation models, and have been shown to contain the majority of the granulocyte/macrophage precursors, NOD/SCID repopulating cells and CD34 + dendritic cell precursors. Phenotypically, CD133 positive cells in blood and marrow are CD34 bright, with CD34 dim CD71 bright cells being negative for CD133 expression. Many leukemias express CD133 as well as CD34 , but some investigators have noted leukemic blasts which are CD133+ and CD34 negative. No natural ligand has yet been demonstrated for the CD133 molecule, and its function in hematopoietic tissue is unknown. However, Maw and colleagues have described a mutation in the CD133 gene, which is responsible for retinal degeneration in an Indian pedigree.

SIZE: 865 amino acids; 97202 Da
SUBCELLULAR LOCATION: Membrane; Multi-pass membrane protein (Potential).
TISSUE SPECIFICITY: Selectively expressed on CD34 hematopoietic stem and progenitor cells in adult and fetal bone marrow, fetal liver, cord blood and adult peripheral blood. Not detected on other blood cells. Also expressed in a number of non-lymphoid tissues including retina, pancreas, placenta, kidney, liver, lung, brain and heart.
PTM: Glycosylated.
DISEASE: SwissProt: O43490 # Defects in PROM1 are the cause of an autosomal recessive form of retinal degeneration [MIM:604365]. The disease is characterized by night blindness and loss of peripheral vision from childhood with progression to profound visual impairment and extinguished electroretinograms by the third decade. A single nucleotide deletion yields a truncated protein lacking half of the second extracellular loop, the final membrane-spanning domain and the cytoplasmic C-terminus.
SIMILARITY: SwissProt: O43490 ## Belongs to the prominin family.

• Mouse Embryonic Stem Cells
• Hematopoietic Stem Cells
• Neural Stem Cells
• Epithelial Cells
• Mesenchymal Stem Cells

Characterization

Chemicon

Anti-CD133, clone 13A4

1.0 mg/mL

Monoclonal Antibody

100 μg

Purified

Rat