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The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 112 kDa (predicted molecular weight: 112 kDa).
Use a concentration of 1 µg/ml.
Serine/threonine-protein kinase involved in autophagy in response to starvation. Acts upstream of phosphatidylinositol 3-kinase PIK3C3 to regulate the formation of autophagophores, the precursors of autophagosomes. Part of regulatory feedback loops in autophagy: acts both as a downstream effector and negative regulator of mammalian target of rapamycin complex 1 (mTORC1) via interaction with RPTOR. Activated via phosphorylation by AMPK and also acts as a regulator of AMPK by mediating phosphorylation of AMPK subunits PRKAA1, PRKAB2 and PRKAG1, leading to negatively regulate AMPK activity. May phosphorylate ATG13/KIAA0652 and RPTOR; however such data need additional evidences. Plays a role early in neuronal differentiation and is required for granule cell axon formation. May also phosphorylate SESN2 and SQSTM1 to regulate autophagy (PubMed:25040165).
Ubiquitously expressed. Detected in the following adult tissues: skeletal muscle, heart, pancreas, brain, placenta, liver, kidney, and lung.
Belongs to the protein kinase superfamily. Ser/Thr protein kinase family. APG1/unc-51/ULK1 subfamily. Contains 1 protein kinase domain.
Autophosphorylated. Phosphorylated under nutrient-rich conditions; dephosphorylated during starvation or following treatment with rapamycin. Under nutrient sufficiency, phosphorylated by MTOR/mTOR, disrupting the interaction with AMPK and preventing activation of ULK1 (By similarity). In response to nutrient limitation, phosphorylated and activated by AMPK, leading to activate autophagy.
Cytoplasm, cytosol. Preautophagosomal structure. Under starvation conditions, is localized to puncate structures primarily representing the isolation membrane that sequesters a portion of the cytoplasm resulting in the formation of an autophagosome.
Western blot - Anti-ULK1 (phospho S757) antibody (ab156920)
All lanes : Anti-ULK1 (phospho S758) antibody (ab156920) at 1 µg/ml
Lane 1 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 : EGF-Stimulated A431 Whole Cell Lysate Lane 3 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate with Immunising peptide at 1 µg/ml Lane 4 : EGF-Stimulated A431 Whole Cell Lysate with Immunising peptide at 1 µg/ml Lane 5 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate with Control peptide at 1 µg/ml Lane 6 : EGF-Stimulated A431 Whole Cell Lysate with Control peptide at 1 µg/ml
Lysates/proteins at 10 µg per lane.
Secondary All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 112 kDa Observed band size: 112 kDa Additional bands at: 130 kDa (possible non-specific binding)
Exposure time: 8 minutes
This blot was produced using a 10% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab156920 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.
ab156920 was tested using an Indirect ELISA approach. The wells were coated with peptide (1µg/ml at 100µl/well) overnight at 4°C, followed by a 5% BSA blocking step for 1 hour at room temperature. The primary Ab was then added at a dilution range of 1- 0.00025µg/ml (100µl/well) for 1hr at room temperature. A HRP-conjugated anti-rabbit IgG (heavy and light chain) was used as a secondary antibody at 1:20,000 dilution for 1hr at room temperature.