The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/2000 - 1/10000. Detects a band of approximately 40 kDa (predicted molecular weight: 40 kDa).
Use at 2-5 µg/mg of lysate.
Subunit of mTORC1, which regulates cell growth and survival in response to nutrient and hormonal signals. mTORC1 is activated in response to growth factors or amino-acids. Growth factor-stimulated mTORC1 activation involves a AKT1-mediated phosphorylation of TSC1-TSC2, which leads to the activation of the RHEB GTPase that potently activates the protein kinase activity of mTORC1. Amino-acid-signaling to mTORC1 requires its relocalization to the lysosomes mediated by the Ragulator complex and the Rag GTPases. Activated mTORC1 up-regulates protein synthesis by phosphorylating key regulators of mRNA translation and ribosome synthesis. mTORC1 phosphorylates EIF4EBP1 and releases it from inhibiting the elongation initiation factor 4E (eiF4E). mTORC1 phosphorylates and activates S6K1 at 'Thr-389', which then promotes protein synthesis by phosphorylating PDCD4 and targeting it for degradation. Within mTORC1, AKT1S1 negatively regulates mTOR activity in a manner that is dependent on its phosphorylation state and binding to 14-3-3 proteins. Inhibits RHEB-GTP-dependent mTORC1 activation. Substrate for AKT1 phosphorylation, but can also be activated by AKT1-independent mechanisms. May also play a role in nerve growth factor-mediated neuroprotection.
Widely expressed with highest levels of expression in liver and heart. Expressed at higher levels in cancer cell lines (e.g. A549 and HeLa) than in normal cell lines (e.g. HEK293).
Phosphorylated by AKT1. Phosphorylation relieves inhibitory function on mTORC1.
Cytoplasm > cytosol. Found in the cytosolic fraction of the brain.
All lanes : Anti-PRAS40 antibody (ab72321) at 0.04 µg/ml
Lane 1 : HeLa whole cell lysate at 50 µg Lane 2 : HeLa whole cell lysate at 15 µg Lane 3 : HeLa whole cell lysate at 5 µg Lane 4 : 293T whole cell lysate at 50 µg
Predicted band size: 40 kDa Observed band size: 40 kDa Additional bands at: 30 kDa, 50 kDa. We are unsure as to the identity of these extra bands.
Exposure time: 30 seconds
Immunoprecipitation - PRAS40 antibody (ab72321)
Detection of PRAS40 by Western Blot of Immunprecipitate.
ab72321 at 1µg/ml for WB, staining PRAS40 in HeLa whole cell lysate subsequent to immunoprecipitation using ab72321 at 3µg/mg lysate (1 mg/IP; 20% of IP loaded/lane). Detection: Chemiluminescence with exposure time of 10 seconds.