Western blot analysis of extracts from THP-1 and HL-60 cells, untreated (-) or treated overnight with TPA #9905 (+), using ASM Antibody.Learn more about how we get our images.
Western blot analysis of extracts from COS-7 cells, untransfected (-) or transfected with a human ASM construct (+), using ASM Antibody.Learn more about how we get our images.
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.
ASM Antibody detects endogenous levels of total human ASM protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues at the carboxyl terminus of human ASM. Antibody was purified by protein A and peptide affinity chromatography.
Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin to produce ceramide and phosphocholine (1). Ceramide is an important bioactive lipid triggering signal transduction involved in cell proliferation, apoptosis and differentiation (1,2). A number of SMases have been described and categorized based on their optimum pH activity, cation dependence, tissue distribution, and subcellular localization (1). These include a lysosomal acid SMase, a Zn++-dependent secreted acid SMase, a membrane-bound Mg++-dependent neutral SMase, a Mg++-independent neutral SMase, and an alkaline SMase.
Acid sphingomyelinase (ASM or SMPD1) is a lysosomal enzyme responsible for the hydrolysis of sphingomyelin to ceramide and phosphocholine. The ASM gene encodes three proteins, ASM-1, ASM-2, and ASM-3, of which ASM-1 is the only catalytically active enzyme (3,4). ASM-1 can exist as a 70 kDa form as well as a 57 kDa proteoytic product (5). Expression of ASM is induced during monocytic cell differentiation (6). Defects in the ASM gene are associated with type A and type B Niemann-Pick disease (7).
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