Western blot analysis of extracts from THP1 cells, untreated or PMA-treated, and guinea pig neutrophils, untreated or fMLP-treated, using Phospho-p40phox (Thr154) Antibody.
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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-p40phox (Thr154) Antibody detects endogenous levels of p40phox only when phosphorylated at threonine 154. This antibody does not cross-react with other phosphorylated phox subunits.Species Reactivity:
Human, Mouse, Guinea Pig
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr154 of human p40phox. Antibodies are purified by protein A and peptide affinity chromatography.
The phagocytic NADPH oxidase is a multiprotein enzyme that catalyzes the reduction of oxygen to superoxide in response to invasion of pathogens into the body. The NADPH oxidase consists of 6 subunits, the membrane-bound p91phox and p22phox heterodimer (also known as cytochrome b558), the cytoplasmic complex of p40phox, p47phox and p67phox, and the small GTPase Rac2. Activation of NADPH oxidase is initiated by phosphorylation of the cytosolic complex, which induces comformational changes of the complex and ultimately leads to the translocation of the cytoplasmic complex to the membrane to form an active enzyme with cytochrome b558 (1). Thr154 and Ser315 of p40 phox have been identified as PKC phosphorylation sites modified during activation of the phagocyte NADPH oxidase (2).
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