Figure1: Treatment of HeLa cells with Calyculin A accumulates phosphorylation of mTOR at Ser2448, as detected by PathScan® Phospho-mTOR (Ser2448) Sandwich ELISA Kit #7976, but does not affect levels of total mTOR protein detected by PathScan® Total mTOR Sandwich ELISA Kit #7974. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using mTOR (7C10) Rabbit mAb #2983 (left panel) or Phospho-mTOR (Ser2448) Antibody #2971 (right panel) is shown in the bottom figure.
Figure 2: The relationship between protein concentration of lysates from untreated and Calyculin A-treated HeLa cells and the absorbance at 450 nm is shown. After starvation, HeLa cells (85% confluence) were treated with Calyculin A (10 nM) for 10-15 min at 37ºC and then lysed.
|REACTIVITY||H M R Mk|
|Product Includes||Volume||Solution Color|
|mTOR Mouse mAb Coated Microwells||96 tests|
|mTOR Rabbit Detection mAb||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Sealing Tape||2 ea|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Total mTOR Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of mTOR protein. A mTOR mouse antibody has been coated onto the microwells. After incubation with cell lysates, mTOR protein is captured by the coated antibody. Following extensive washing, an mTOR rabbit antibody is added to detect captured mTOR protein. Anti-rabbit IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate TMB is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of mTOR protein.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Total mTOR Sandwich ELISA Kit #7974 detects endogenous levels of total human mTOR as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Human, Mouse, Rat, Monkey
The mammalian target of rapamycin (mTOR, FRAP, RAFT) is a Ser/Thr protein kinase (1-3) that functions as an ATP and amino acid sensor to balance nutrient availability and cell growth (4,5). When sufficient nutrients are available, mTOR responds to a phosphatidic acid-mediated signal to transmit a positive signal to p70 S6 kinase and participate in the inactivation of the eIF4E inhibitor, 4E-BP1 (6). These events result in the translation of specific mRNA subpopulations. mTOR is phosphorylated at Ser2448 via the PI3 kinase/Akt signaling pathway and autophosphorylated at Ser2481 (7,8). mTOR plays a key role in cell growth and homeostasis and may be abnormally regulated in tumors. For these reasons, mTOR is currently under investigation as a potential target for anti-cancer therapy (9).
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