Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total mTOR Sandwich ELISA Kit #7974

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Kit Includes Volume Solution Color
mTOR Mouse Antibody coated microwells 96 tests
mTOR 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
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 Cross-Reactivity

H M R Mk

Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

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.

Specificity / Sensitivity

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.

ELISA - Western correlation

ELISA - Western correlation

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.

Sensitivity

Sensitivity

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.

Background

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).

  1. Sabers, C.J. et al. (1995) J Biol Chem 270, 815-22.
  2. Brown, E.J. et al. (1994) Nature 369, 756-8.
  3. Sabatini, D.M. et al. (1994) Cell 78, 35-43.
  4. Gingras, A.C. et al. (2001) Genes Dev 15, 807-26.
  5. Dennis, P.B. et al. (2001) Science 294, 1102-5.
  6. Fang, Y. et al. (2001) Science 294, 1942-5.
  7. Navé, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.
  8. Peterson, R.T. et al. (2000) J Biol Chem 275, 7416-23.
  9. Huang, S. and Houghton, P.J. (2003) Curr Opin Pharmacol 3, 371-7.

Application References

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For Research Use Only. Not For Use In Diagnostic Procedures.

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