Western blot analysis of extracts from A172 cells, untreated (-), Torin 1-treated (250 nM; 5 hr; +), or INK128-treated (250 nM; 5 hr; +) using Phospho-ULK1 (Ser638) (D8K9O) Rabbit mAb (upper) or ULK1 (D8H5) Rabbit mAb #8054 (lower).
Western blot analysis of A172 cell extracts, untreated (-) or λ phosphatase and calf intestinal phosphatase (CIP)-treated (+), using Phospho-ULK1 (Ser638) (D8K9O) Rabbit mAb (upper) or ULK1 (D8H5) Rabbit mAb #8054 (lower).
Western blot analysis of various cell lines using Phospho-ULK1 (Ser638) (D8K9O) Rabbit mAb.
|REACTIVITY||H M Mk|
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 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
Phospho-ULK1 (Ser638) (D8K9O) Rabbit mAb recognizes endogenous levels of ULK1 protein only when phosphorylated at Ser638.Species Reactivity:
Human, Mouse, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser638 of human ULK1 protein.
Two related serine/threonine kinases, UNC-51-like kinase 1 and 2 (ULK1, ULK2), were discovered as mammalian homologs of the C. elegans gene UNC-51 in which mutants exhibited abnormal axonal extension and growth (1-4). Both proteins are widely expressed and contain an amino-terminal kinase domain followed by a central proline/serine rich domain and a highly conserved carboxy-terminal domain. The roles of ULK1 and ULK2 in axon growth have been linked to studies showing that the kinases are localized to neuronal growth cones and are involved in endocytosis of critical growth factors, such as NGF (5). Yeast two-hybrid studies found ULK1/2 associated with modulators of the endocytic pathway, SynGAP and syntenin (6). Structural similarity of ULK1/2 has also been recognized with the yeast autophagy protein Atg1/Apg1 (7). Knockdown experiments using siRNA demonstrated that ULK1 is essential for autophagy (8), a catabolic process for the degradation of bulk cytoplasmic contents (9,10). It appears that Atg1/ULK1 can act as a convergence point for multiple signals that control autophagy (11), and can bind to several autophagy-related (Atg) proteins, regulating phosphorylation states and protein trafficking (12-16).
Phosphorylation of ULK1 at Ser638 and Ser757 is mediated by mTOR, which is a regulator of cell growth and an inhibitor of autophagy that disrupts the interaction between ULK1 and AMPK (17,18). Conversely, AMPK is activated during low nutrient conditions and directly phosphorylates ULK1 at multiple sites including Ser317, Ser555, and Ser777 (17-19).
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