Sequence:
GPHMSDEEILEKLRSIVSVGDPKKKYTRFEKIGQGASGTVYTAMDVATGQEVAIKQMNLQQQPKKELIINEILVMREN
KNPNIVNYLDSYLVGDELWVVMEYLAGGSLTDVVTETCMDEGQIAAVCRECLQALEFLHSNQVIHRDIKSDNILLGM
DGSVKLTDFGFCAQITPEQSKRSTMVGTPYWMAPEVVTRKAYGPKVDIWSLGIMAIEMIEGEPPYLNENPLRALYLIA
TNGTPELQNPEKLSAIFRDFLNRCLEMDVEKRGSAKELLQHQFLKIAKPLSSLTPLIAAAKEATKNNH
- Underlined amino acids (GPHM) are from HRV 3C cleavage site.
- Phosphorylated at T423.
Formulation:
8 mg/ml in 25 mM Tris-HCl, pH8.0, 150 mM NaCl, 10% glycerol, 5 mM DTT.
Pak1KD Activity:
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Specific activity:
PAK1=32,638 pmoles/min/mg
Analysis of enzymatic activity was performed according to Zlyte assay protocol (Invitrogen):
- Different concentration of PAK1 were incubated in a buffer containing 50 mM HEPES (pH7.5), 10 mM MgCl2, 1 mM EGTA, 200 mM ATP, 0.01% Brij-35, and 2 µM substrate (SER/THR 14, Invitrogen) at room temperature for 1 hour.
- Developer solution was added to reaction and reaction was stopped after 1h of incubation at RT.
- Fluorescence was detected using lexc=460±40 nm and lem=528±20 nm filters.
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SDS-PAGE Analysis:
MASS-Spec Analysis:
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Calculated mass: 33,646
Measured mass: 33,728 (1P), 33,808 (2P), 33,889 (3P), 33,968 (4P)
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