Western blot analysis of extracts from various cell lines using Malic Enzyme 3 (E8A3V) Rabbit mAb.
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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
Malic Enzyme 3 (E8A3V) Rabbit mAb recognizes endogenous levels of total malic enzyme 3 protein. This antibody does not cross-react with malic enzyme 1 and malic enzyme 2 proteins.Species Reactivity:
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala390 of human malic enzyme 3 protein.
Malic enzymes catalyze oxidative decarboxylation of malate to pyruvate (1). The malic enzyme family in mammalian cells includes the cytosolic malic enzyme 1 (ME1) and two mitochondrial malic enzymes (ME2 and ME3) (1, 2). ME1 and ME2 are critical for tumor cell growth and their expression is repressed by tumor suppressor p53 (2). Reduced expression of ME1 and ME2 reciprocally increases the levels and activation of p53, promoting p53-mediated senescence (2). Research studies show ME3 is essential for the survival of pancreatic ductal adenocarcinoma following genomic deletion of ME2 (3). Deletion of ME3 is lethal to ME2-null cancer cells, which has been suggested to provide a potential therapeutic opportunity using collateral lethality (3, 4).
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