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
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.
SOAT1 Antibody recognizes endogenous levels of total SOAT1 protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human SOAT1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
SOAT1 (Sterol O-acyltransferase 1; ACAT1) is an O-acyltransferase that functions in the endoplasmic reticulum (ER) to catalyze the formation of cholesterol esters from free cholesterol and long chain fatty acyl-coenzyme A. The cholesterol esters are incorporated into cytoplasmic lipid droplets, thereby preventing excess free cholesterol from inducing lipid-mediated cell toxicity, including ER stress (1). Research studies have shown that pharmacological inhibition of SOAT1 in tumor cells induced lipid-mediated cell toxicity that suppressed tumor cell growth and promoted tumor cell apoptosis (2-3). Pharmacological SOAT1 inhibition was also shown to stimulate autophagy-mediated proteolysis in microglia, leading to enhanced clearance of amyloid peptide Aβ42 (4, 5). Collectively, these findings suggest that SOAT1 inhibition may have therapeutic potential in both cancer and Alzheimer’s disease.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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