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EMD Chemicals, Inc.
480 S. Democrat Road
08027 Gibbstown
United States
Phone: 800 854 3417
Fax: 800 776 0999
E-mail: emdinfo@emdchemicals.com
30 July 2010
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The K-LISA™ Akt Activity Kit is a rapid, sensitive, ELISA-based activity assay suitable for measuring the kinase activity of purified or partially purified Akt preparations, in vitro Akt inhibitor screening, and for assessing the regulation of Akt in cell signaling. The assay utilizes a biotinylated peptide substrate (GRPRTSSFAEG) that is phosphorylated on the second serine by Akt1, Akt2, Akt3, SGK (Serum Glucocorticoid Kinase), and MSK1. Biotinylated Akt substrate and sample containing Akt are incubated in the presence of ATP in wells of a streptavidin-coated 96-well plate, which allows for phosphorylation and substrate capture in a single step.
| Product information | ||||
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| Format | 96-well plate | |||
| Form | 96 Tests | |||
| Detection method | Colorimetric | |||
| Unit definition | One unit is defined to the amount of enzyme required to transfer 1 nmol phosphate per min to substrate at 30° C. | |||
| Components |
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| Sensitivity | 0.05 mUnits | |||
| Assay range | 6-100 mU/well | |||
| Assay time | 3 h | |||
| Sample type | Cell lysates, tissue extracts, purified enzyme | |||
| Kit contains | Streptavidin-Coated 96-Well Plate, Akt Substrate, Biotinylated, Phosphoserine Detection Antibody, Antibody Diluent, Kinase Assay Buffer, 5X ATP/MgCl2 Mix, 100X Inhibitor, Kinase Stop Solution, ELISA 20X Plate Wash Concentrate, HRP-Antibody Conjugate, TMB Substrate, ELISA Stop Solution, Plate Sealers, and a user protocol. Sold under license of U.S. patent 6,441,140. Manufactured by Cell Signaling. | |||
| Declaration | *Sold under license of U.S. patent 6,441,140 Manufactured by Cell Signaling | |||
| Store and ship information | |||
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<b>Hazardous Materials Attention:</b> Due to the nature of the Hazardous Materials in this shipment, additional shipping charges <i>may</i> be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges. |
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| Storage | -20°C | ||
| Ship |
Dry Ice Only
Multiple Toxicity Values, refer to MSDS |
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| Data | |||
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 Near-confluent MCF-7 cells were stimulated with IGF-1 (100 ng/ml) or IGF-1 (100 ng/ml) and 17β-estradiol (500 nM) for 30 min at 37°C in a CO<sub>2</sub> incubator. For inhibition of Akt, cells were first pre-incubated at 37°C in a CO<sub>2</sub> incubator for 15 min in the presence of cell-permeable Akt Inhibitor II (Cat. No. 124008) followed by stimulation with IGF-1 (100 ng/ml) for 30 min at 37°C. Cell lysates were prepared using 0.5 ml PhosphoSafe™ Extraction Buffer per 10 cm cell culture dish, per the standard protocol. Cell lysates were either used immediately or stored at -70°C until use. Equal amounts of total protein (1.5 mg) were immunoprecipitated and the activity was determined as outlined in protocol B under the Detailed Protocol section. |
