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Akt1/Protein Kinase B |
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Description:
Akt1/PKB is the focal point for survival signals from growth and survival factor receptors, transduced via P13-kinase pathway. Theonine 308 and serine 473 of AKT is phosphorylated by PIP3 and PDK2 respectively. AKT promotes cell survival directly by means of its ability to phosphorylate and inactivate several pro-apoptotic targets.
Source:
Over expressed as a MBP-Akt1 fusion in Spodoptera frugiperda (Sf9) cells infected with a recombinant baculovirus encoding Akt1 residues 140-480 with T308D and S473D mutation.
Reagents Supplied:
Akt1/PKB Reaction Buffer (10X)
Enzyme Properties
Protein Kinase Substrate Recognition
Molecular Weight:
Theoretical: 79,000 daltons
Specific Activity:
30,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X Akt1/PKB Reaction Buffer
Supplemented with 200 μM ATP
Incubate at
30°C.
1X Akt1/PKB Reaction Buffer:
20 mM Tris-HCl
10 mM MgCl2
5 mM DTT
pH 7.5 @ 25°C
Unit Definition:
One unit is defined as the amount of Akt1/PKB required to catalyze the transfer of 1 pmol of phosphate to GRPRTSSFAEGKK (30 µM) in 1 minute at 30°C in a total reaction volume of 25 µl.
Note that optimal times and enzyme concentrations must be determined empirically for each particular substrate.
Storage Conditions:
25 mM Tris-HCl
50 mM NaCl
1 mM Dithiothreitol
1 mM EDTA
50% Glycerol
0.03% Brij 35
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Purified Akt1/PKB contains no detectable protease or phosphatase activities.
Protease Activity:
After incubation of 500 units of Akt1/Protein Kinase B with a standard mixture of proteins
for 2 hours at 30ºC, no proteolytic activity could be detected by SDS-PAGE.
Contaminating Phosphatases:
After incubation of 500 units of with 50 mM p-nitrophenyl phosphate for 2 hours at 30ºC, no
phosphatase activity could be detected by spectrophotometric analysis.
References
- Cohen, P. et al. (1997) FEBS Lett., 410, 3-10.
- Alessi, D.R. and Cohen, P. (1988) Curr. Opin. Genet. Dev., 8, 55-62.
Reagents Sold Separately
Akt1/PKB Reaction Buffer
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