Western blot analysis of extracts from COS cells, untransfected, or transfected with either 5-LO wild type or GFP 5-LO (Ser663Ala), using Phospho-5-Lipoxygenase (Ser663) Antibody (upper) and 5-Lipoxygenase (C49G1) Rabbit mAb #3289 (lower). (We are thankful to Dr. Flamand from University of Michigan for providing the overexpression constructs and for his help in characterizing this antibody).
Western blot analysis of extracts from COS cells, transfected with 5-LO wild type or GFP 5-LO (Ser663Ala), using Phospho-5-Lipoxygenase (Ser663) Antibody. The phospho-specificity of the antibody was verified by preincubating the antibody with no peptide (left), with 5-LO (Ser663) non-phosphopeptide (middle) or 5-LO (Ser663) phosphopeptide (right) prior to incubating the membrane.
|MW (kDa)||78, 80|
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
Phospho-5-Lipoxygenase (Ser663) Antibody detects overexpressed phospho-5-lipoxygenase protein only when phosphorylated at Ser663.Species Reactivity:
Antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser663 of human 5-lipoxygenase protein.
5-Lipoxygenase (5-LO, ALOX5) is an important catalytic enzyme responsible for the biosynthesis of leukotriene LTA4 from arachidonic acid (1,2). Leukotriene synthesis also requires 5-lipoxygenase-activating protein (FLAP, ALOX5AP), a nuclear membrane-bound protein that binds arachidonic acid and is thought to activate 5-LO. A number of related leukotrienes (i.e. B4, C4, D4) are derived from LTA4 and together these lipid mediators function in immune reaction regulation. 5-LO is primarily expressed in polymorphonuclear leukocytes, peripheral blood monocytes, macrophages, and mast cells (1,3). Overexpression of 5-LO protein is seen in certain cancer cells and is associated with poor diagnosis (1,4). Depending upon the cell type, 5-LO is localized to either the cytosol or the nucleus of quiescent cells (5). Following stimulation, 5-LO translocates to the nucleus and associates with FLAP to catalyze LTA4 synthesis (2,3). Phosphorylation of specific residues can regulate 5-LO enzymatic activity. Phosphorylation of 5-LO at Ser523 by PKA family kinases inhibits oxygenase activity (6,7) while MAPKAP2 and ERK family kinase phosphorylation at Ser271 and Ser663 stimulates 5-LO enzymatic activity in vivo (8,9).
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