Western blot analysis of extracts from SH-SY5Y cells, untreated (-) or treated with TPA (200 nM, 24 hr) #4174 (+), and 293 cells, using Dopamine β-Hydroxylase (DBH) 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
Dopamine β-Hydroxylase (DBH) Antibody recognizes endogenous levels of total DBH protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala211 of human dopamine β-hydroxylase protein. Antibodies are purified by protein A and peptide affinity chromatography.
Dopamine β-Hydroxylase (DBH) is an enzyme of the copper type II ascorbate-dependent mono-oxygenase family. This enzyme forms homotetramers composed of two noncovalently bound disulfide-linked dimers and is found as both membrane-associated and soluble forms (1-3). The soluble form is present in the lumen of secretory granules (4) and is released from cells by exocytosis (5). DBH converts dopamine to noradrenaline (6). Deficiency in this enzyme causes a rare disease characterized by a complete absence of noradrenaline and adrenaline in plasma together with increased plasma dopamine levels (7). Orthostatic hypotension, the main symptom of DBH deficiency, can be alleviated by administration of dihydroxyphenylserine, a synthetic precursor of noradrenaline (8).
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