Western blot analysis of extracts from differentiated NIH/3T3-L1 cells treated with isoproterenol, using Phospho-HSL (Ser660) Antibody.
|MW (kDa)||81, 83|
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 nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody 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 June 2020
Protocol Id: 263
Phospho-HSL (Ser660) Antibody detects endogenous levels of HSL only when phosphorylated at Ser660 by PKA. This antibody does not cross-react with Ser563, Ser565 or Ser659 phosphorylated HSL.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser651 of mouse HSL (equivalent to Ser660 of rat HSL). Antibodies are purified by protein A and peptide affinity chromatography.
HSL (hormone-sensitive lipase) catalyzes the hydrolysis of triacylglycerol, the rate-limiting step in lipolysis. Lipolytic stimuli activate adenylyl cyclase and thus increase intracellular cAMP levels, which in turn activate protein kinase A (PKA). PKA phosphorylates HSL at Ser563, Ser659, and Ser660, which stimulates HSL activity (1,2). In contrast, AMPK phosphorylates HSL at Ser565, which reduces HSL phosphorylation at Ser563 by PKA and inhibits HSL activity (2,3). Recent work indicates that phosphorylation at Ser600 by p44/42 MAPKs also enhances the enzymatic activity of HSL (4).
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