Western blot analysis of extracts from various cell lines using IL3RA/CD123 Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 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.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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 October 2016
Protocol Id: 10
IL3RA/CD123 Antibody recognizes endogenous levels of total IL3RA/CD123 protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human IL3RA/CD123 protein. Antibodies are purified by peptide affinity chromatography.
IL3RA/CD123 is the alpha subunit of an IL-3 receptor that also contains the common beta chain (βc) shared with receptors for other cytokines. IL3RA/CD123 alone binds to IL-3 with high specificity but low affinity and it does not transduce intracellular signaling, while βc alone does not bind to IL-3 appreciably. Together, IL3RA/CD123 and βc form the functional high-affinity receptor for IL-3. IL3RA/CD123 is mainly expressed on the hematopoietic progenitor cells, primarily the granulocytic and monocytic linages (1,2). IL3RA/CD123 is highly expressed on leukemic stem cells in the majority of AML (acute myeloid leukemia) patients and is correlated with poor prognosis. In addition, overexpression of IL3RA/CD123 has also been reported for other hematopoietic malignancies (3-5). It is considered as a valuable therapeutic target for these malignancies (6-8).
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