Product Pathways - PathScan ELISA
PathScan® Phospho-mTOR (Ser2481) Sandwich ELISA Kit #7978
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|Kit Includes||Volume||Solution Color|
|mTOR Mouse Antibody coated microwells||96 tests|
|Phospho-mTOR (Ser2481) Rabbit Detection Antibody||11 ml||Green|
|Anti-rabbit IgG, HRP-linked Antibody||11 ml||Red|
|TMB Substrate #7004||11 ml||Colorless|
|STOP Solution #7002||11 ml||Colorless|
|Sealing Tape||2 sheets|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) #9803||15 ml||Yellowish|
Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).
Species enclosed in parentheses are predicted to react based on 100% sequence homology.
The PathScan® Phospho-mTOR (Ser2481) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of mTOR protein phosphorylated at Ser2481. A mTOR Mouse mAb has been coated onto the microwells. After incubation with cell lysates, mTOR (phospho and nonphospho) protein is captured by the coated antibody. Following extensive washing, Phospho-mTOR (Ser2481) Rabbit mAb is added to detect the captured phospho-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 phosphorylated at Ser2481.Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
PathScan® Phospho-mTOR (Ser2481) Sandwich ELISA Kit #7978 detects endogenous levels of human mTOR protein when phosphorylated at Ser2481 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. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
ELISA - Western correlation
Figure 1. Treatment of HeLa cells with Calyculin A #9902 accumulates phosphorylation of mTOR at Ser2448 or Ser2481, as detected by PathScan® Phospho-mTOR (Ser2448) Sandwich ELISA Kit #7976 or PathScan® Phospho-mTOR (Ser2481) Sandwich ELISA Kit #7978, 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, Phospho-mTOR (Ser2448) Antibody #2971 or Phospho-mTOR (Ser2481) Antibody #2974 is shown in the bottom figure.
Figure 2. The relationship between protein concentration of lysates from HeLa cells, untreated or reated with Calyculin A #9902, and the absorbance at 450 nm is shown. After starvation, HeLa cells (85% confluence) were treated with Calyculin A #9902 (10 nM) for 10-15 min at 37ºC and then lysed.
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).
- Sabers, C.J. et al. (1995) J Biol Chem 270, 815-22.
- Brown, E.J. et al. (1994) Nature 369, 756-8.
- Sabatini, D.M. et al. (1994) Cell 78, 35-43.
- Gingras, A.C. et al. (2001) Genes Dev 15, 807-26.
- Dennis, P.B. et al. (2001) Science 294, 1102-5.
- Fang, Y. et al. (2001) Science 294, 1942-5.
- Navé, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.
- Peterson, R.T. et al. (2000) J Biol Chem 275, 7416-23.
- Huang, S. and Houghton, P.J. (2003) Curr Opin Pharmacol 3, 371-7.
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- 7974 PathScan® Total mTOR Sandwich ELISA Kit
- 7976 PathScan® Phospho-mTOR (Ser2448) Sandwich ELISA Kit
- 2972 mTOR Antibody
- 4517 mTOR (L27D4) Mouse mAb
- 2983 mTOR (7C10) Rabbit mAb
- 2971 Phospho-mTOR (Ser2448) Antibody
- 2974 Phospho-mTOR (Ser2481) Antibody
- 5536 Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 9902 Calyculin A
- 9803 Cell Lysis Buffer (10X)
- 7004 TMB Substrate
- 7002 STOP Solution
- 9808 Phosphate Buffered Saline (PBS-20X)
- 9809 Phosphate Buffered Saline with Tween 20 (PBST-20X)
For Research Use Only. Not For Use In Diagnostic Procedures.