Product Pathways - PathScan ELISA
PathScan® Total Akt1 Sandwich ELISA Kit #7170
|7170C||1 Kit (96 assays)||---||In Stock||---|
|7170||carrier free and custom formulation / quantity||email request|
When ordering five or more kits, please contact us for processing time and pricing at email@example.com.
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|Kit Includes||Volume||Solution Color|
|Akt Rabbit mAb coated microwells||96 tests|
|Akt1 Mouse Detection mAb||1 Ea||Green (Lyophilized)|
|Anti-mouse 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||Colorless|
|STOP Solution #7002||11 ml||Colorless|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|Sealing Tape||2 sheets|
|Cell Lysis Buffer (10X) #9803||15 ml||Yellowish|
Note: 12 8-well modules – Each module is designed to break apart for 8 tests.
Storage: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).
CST's PathScan® Total Akt1 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total Akt1 protein. An Akt Antibody has been coated onto the microwells. After incubation with cell lysates, the Akt protein is captured by the coated antibody. Following extensive washing, Akt1 Mouse Monoclonal Antibody is added to detect the captured total Akt1 protein. Anti-Mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of total Akt1 protein.
Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Total Akt1 Sandwich ELISA Kit detects endogenous levels of Akt1 including phosphorylated and nonphosphorylated. Inhibition of phosphorylation of Akt at serine 473 by LY294002 in NIH/3T3 cells is detected by PathScan Phospho-Akt1 (Ser473) Sandwich ELISA kit , #7160, while the level of total Akt1 protein detected by this ELISA kit #7170 is unchanged, as seen in Figure 1. Similar results have also observed in Jurkat cells (data not shown). This ELISA kit does not detect related kinases, such as PKC and p70 S6 kinase. 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 NIH/3T3 cells with various concentrations of LY294002 blocks PDGF-stimulated phosphorylation of Akt1 at serine 473 detected by PathScan Phospho-Akt1 (Ser473) Sandwich ELISA kit , #7160, but does not affect the level of total Akt1 protein detected by PathScan Total Akt1 Sandwich ELISA kit #7170. OD450 readings are shown in the top figure, while the corresponding Western blot, using Phospho-Akt (Ser473) Antibody #9271 or Akt Antibody #9272, is shown in the bottom figure.
Figure 4: Linear relationship between protein concentration of lysates from either control NIH/3T3 cells (dotted line) or PDGF-treated NIH/3T3 cells (solid line) and kit assay optical density readings.
Figure 3: Comparison of sensitivity between sandwich ELISA and Western blot in the detection of recombinant nonphospho-Akt1 protein, using Akt Antibody #9272. The top figure indicates that, at 0-1,000 pg of protein, the concentration of nonphospho-Akt1 is linearly proportional to the OD450 reading from the Sandwich ELISA kit.
Figure 2: Comparison of sensitivity between sandwich ELISA and Western blot in the detection of recombinant phospho-Akt1 protein, using Akt Antibody #9272. The top figure indicates that, at 0-500 pg of protein, the concentration of phospho-Akt1 is linearly proportional to the OD450 reading from the Sandwich ELISA kit.
Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (5,6). Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including Bad (7), forkhead transcription factors (8), c-Raf (9), and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). LY294002 is a specific PI3 kinase inhibitor (11). Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β (12,13). Akt may also play a role in insulin stimulation of glucose transport (12). In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1 (14) and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 (15) and p21 Waf1/Cip1 (16). Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor (17). More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex (18,19).
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- 7160 PathScan® Phospho-Akt1 (Ser473) Sandwich ELISA Kit
- 4060 Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb
- 9271 Phospho-Akt (Ser473) Antibody
- 4051 Phospho-Akt (Ser473) (587F11) Mouse mAb
- 2938 Akt1 (C73H10) Rabbit mAb
- 2967 Akt1 (2H10) Mouse mAb
- 9272 Akt Antibody
- 7076 Anti-mouse IgG, HRP-linked Antibody
- 9901 LY294002
- 9803 Cell Lysis Buffer (10X)
- 7004 TMB Substrate
- 7002 STOP Solution
For Research Use Only. Not For Use In Diagnostic Procedures.
PathScan® is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.