Western blot analysis of extracts from human spinal cord, mouse spinal cord, and mouse kidney using ChAT (E4F9G) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
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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
ChAT (E4F9G) Rabbit mAb recognizes endogenous levels of total human, mouse, and rat ChAT protein.Species Reactivity:
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the carboxy terminus of human ChAT protein.
Choline O-acetyltransferase (ChAT) catalyzes the synthesis of the neurotransmitter acetylcholine (ACh) from acetyl CoA and choline in the central and peripheral nervous system (1). In mammals, ACh is the primary neurotransmitter used at the neuromuscular junction, released by motor neurons to activate muscles. Neurons that release ACh are known as cholinergic neurons, which express high levels of ChAT. In addition to motor neurons, cholinergic neurons are present in the brain, particularly in the basal forebrain as well as other regions of the brain, including striatal interneurons. Reduction in ChAT, indicative of degeneration of cholinergic neurons, is correlated with cognitive decline in several neurodegenerative diseases, including Alzheimer’s disease (2).
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