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Author Hill, Michelle M. ♦ Feng, Jianhua ♦ Hemmings, Brian A.
Source CiteSeerX
Content type Text
File Format PDF
Subject Domain (in DDC) Computer science, information & general works ♦ Data processing & computer science
Subject Keyword Partially Purified Ser473 ♦ Staurosporine Sensitivity ♦ Figure S2 ♦ Kinase Reaction ♦ Pkb Protein ♦ Right Panel ♦ Pooled Superdex-200 Peak Activity Fraction ♦ Immunoprecipitated Pkb Protein ♦ Relative Amount ♦ Top Panel ♦ Phospho-ser473-specific Antibody ♦ Ha-tagged Pkb ♦ Overnight Starvation ♦ Left Panel ♦ Np-40 Lysis Buffer S1 ♦ Coomassie Blue ♦ Immunoglobulin Heavy Chain ♦ Kinase Buffer ♦ Ha-tagged Pkb Isoforms ♦ Np-40 Lysis Buffer ♦ Ser473 Phosphorylation ♦ Sds-page Sample Buffer
Abstract HA-tagged PKB isoforms and mutants were individually transfected into HEK 293 cells. After overnight starvation, lysates were prepared using NP-40 lysis buffer [S1]. HA-tagged PKB was immunoprecipitated and dephosphorylated with-phosphatase (CST). Beads were washed as described previously and were then incubated in kinase buffer alone (left panel) or with pooled Superdex-200 peak activity fractions (right panel) for 60 min at 30C. After the kinase reactions, beads were washed two times in NP-40 lysis buffer and were then heated at 95C in SDS-PAGE sample buffer. Ser473 phosphorylation of the immunoprecipitated PKB proteins was detected by immunoblotting with phospho-Ser473-specific antibodies (top panel). The membrane was stained with Coomassie blue to show the relative amounts of PKB proteins present in the kinase reactions (lower panel). The position of PKB is marked with arrowheads. Immunoglobulin heavy chain can be seen across all lanes.
Educational Role Student ♦ Teacher
Age Range above 22 year
Educational Use Research
Education Level UG and PG ♦ Career/Technical Study