Western blot analysis of extracts from various cell lines using CD133 (A3G6K) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb (lower).
Western blot analysis of extracts from WERI-Rb-1 cells, untreated (-) or treated with PNGase F (+) using CD133 (A3G6K) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
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.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 263
CD133 (A3G6K) Rabbit mAb recognizes endogenous levels of total CD133 protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp562 of human CD133.
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).
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