Western blot analysis of extracts from various cells using Puma Antibody.
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
Puma Antibody detects endogenous levels of total Puma protein. The antibody also cross-reacts with an 18kDa band of unknown origin.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the carboxy-terminal region of human Puma. Antibodies are purified by protein A and peptide affinity chromatography.
Puma (p53 upregulated modulator of apoptosis) is a "BH3-only" Bcl-2 family member originally identified in differential gene expression studies as a p53-inducible gene (1,2). The "BH3-only" family members include Bad, Bid, Bik, Hrk, Bim, and Noxa, all of which contain a BH3 domain but lack other conserved domains, BH1 and BH2, and generally promote apoptosis by binding to and antagonizing anti-apoptotic Bcl-2 family members through BH3 domain interactions (3). Two BH3-containing proteins are produced from the puma gene, Puma-α and Puma-β, both of which are induced by p53, bind Bcl-2 and Bcl-xL, localize to the mitochondria, and promote cytochrome c release and apoptosis (1,2). Puma plays a critical role in the p53 tumor suppressor pathway. Targeted disruption of the puma gene impairs p53-mediated apoptosis and tumor suppression (4-7). Puma knockout mice show defects from multiple apoptotic stimuli, including ionizing irradiation, deregulated c-Myc expression, and cytokine withdrawal (4).
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