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60577
CD133 (A8N6N) Mouse mAb (Flow Specific)
Primary Antibodies
Monoclonal Antibody

CD133 (A8N6N) Mouse mAb (Flow Specific) #60577

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Flow Cytometry Image 1: CD133 (A8N6N) Mouse mAb (Flow Specific)

Flow cytometric analysis of live 293T cells (blue) and Weri-Rb1 cells (green) using CD133 (A8N6N) Mouse mAb (Flow Specific) (solid lines) or a concentration-matched Mouse (G3A1) mAb IgG1 Isotype Control #5415 (dashed lines). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.

To Purchase # 60577S
Product # Size Price
60577S
100 µl N/A

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Mouse IgG3

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Usage Information

Application Dilution
Flow Cytometry 1:50 - 1:100

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Protocol

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Flow Cytometry, Live Cell Protocol for Unconjugated Mouse Antibodies

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
  2. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
  3. Recommended Anti-Mouse secondary antibodies::
    • Anti-Mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408
    • Anti-Mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) #8890
    • Anti-Mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4410
    • Anti-Mouse IgG (H+L), F(ab')2 Fragment (PE Conjugate) #59997

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.

B. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to Immunostaining.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  2. Pellet cells by centrifugation and remove supernatant.
  3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 30 min to 1 hr on ice. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in Antibody Dilution Buffer at the recommended dilution).
  7. Incubate for 30 min on ice. Protect from light.
  8. Wash by centrifugation in Antibody Dilution Buffer. Discard supernatant. Repeat.
  9. Resuspend cells in 200-500 µl of Antibody Dilution Buffer and analyze on flow cytometer.

posted February 2011

revised June 2020

Protocol Id: 58

Specificity / Sensitivity

CD133 (A8N6N) Mouse mAb (Flow Specific) recognizes endogenous levels of total CD133 protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with cells overexpressing human CD133 protein.

Background

CD133, also known as Prominin, was first described as a cell surface marker recognized by monoclonal antibody AC133 on putative hematopoietic stem cells (1). Subsequent cDNA cloning indicated that CD133 is a five-transmembrane protein with a predicated molecular weight of 97 kDa. Due to heavy glycosylation, its apparent molecular weight is 130 kDa as determined by SDS-PAGE analysis (2). Besides blood stem cells, CD133 is expressed on and used to isolate other stem cells, including cancer stem cells (3-7). A deletion mutation in CD133 produces aberrant protein localization and may result in retinal degeneration in humans (8).

  1. Yin, A.H. et al. (1997) Blood 90, 5002-12.
  2. Miraglia, S. et al. (1997) Blood 90, 5013-21.
  3. Handgretinger, R. et al. (2003) Ann N Y Acad Sci 996, 141-51.
  4. Monzani, E. et al. (2007) Eur J Cancer 43, 935-46.
  5. O'Brien, C.A. et al. (2007) Nature 445, 106-10.
  6. Ricci-Vitiani, L. et al. (2007) Nature 445, 111-5.
  7. Singh, S.K. et al. (2004) Nature 432, 396-401.
  8. Maw, M.A. et al. (2000) Hum. Mol. Genet. 9, 27-34.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

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For Research Use Only. Not For Use In Diagnostic Procedures.
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