Used in the Immunoprecipitation and assessment of Akt 1 kinase activity from lysate. The sequence of this peptide (RPRAATF) is related to the sequence surrounding the
phosphorylation site of Glycogen Synthase Kinase-3 (GSK3). This peptide, which has one potential phosphorylation site, is a relatively specific substrate for Akt/PKB, because it is not phosphorylated by other protein kinases such as Rsk-2 and p70 S6 Kinase. The
peptide can be used to assay the activity of immunoprecipitated Akt/PKB.
Capacity: 25 assays
Components:
5X ADBI
Anti-Akt/PKB PH Domain
Akt/SGK Speci?c Peptide
PKA Inhibitor Peptide
Mg/ATP Cocktai
p81 Paper
Other components required but not included as part of kit are:
Enzyme preparation or stimulated cell extract-containing active Akt/PKB
Protein G Agarose
Buffer A: 50mM Tris, pH 7.5, 1mM EDTA, 1mM EGTA, 0.5mM NaVO4, 0.1% (v/v) 2-mercaptoethanol, 1% Triton X-100, 50mM sodium fluoride, 5mM sodium pyrophosphate, 10mM sodium -glycerol phosphate, 0.1mM PMSF, 1ug/ml of aprotinin, pepstatin, leupeptin, and 1uM Microcystin
Buffer B: 50mM Tris-HCl, pH 7.5, 0.03% (w/v) Brij-35, 0.1mM EGTA and 0.1% (v/v) 2-mercaptoethanol
acetone
vortex mixer
plexiglass shielding
shaking incubator
timer
variable volume (5-200ul) pipet + tips
phosphoric acid
scintillation vials
scintillation fluid
scintillation counter
[g-32P]ATP- ~3000 Ci/mmol
Storage and Stability:
Stable for 1 year at -20 degrees C from date of shipment.
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