C8 sep pak spe columns

In vitro phosphorylation of FGF14 was performed using recombinant GSK–3β expressed from a rabbit skeletal muscle cDNA library (Cat # P6040S) with methods from New England Biolabs. Briefly, 98 nmoles (25 µL of a 7.5 mg/mL stock solution) of FGF14 peptide [KPGVTPSKSTSASAIMNGGK-NH₂] were incubated with 1× reaction buffer and 10,000 units (20 µL) of GSK–3 enzyme, and incubated for 2 h at 37 °C. Control studies were performed under identical conditions, except that experiments lacked the addition of the kinase to the reaction solution. Subsequent reduction of the peptide was performed using 10 mM DTT for 30 min at RT. The peptide was alkylated using 5 mM IAA (30 min at RT) and digested with sequencing grade Glu-C 1:00 (w/w) overnight at 37 °C. The digested samples were desalted using C₈ Sep-Pak SPE columns (Waters, Milford, MA) and the eluent was lyophilized in vacuo.

Glycogen Synthase Kinase 3 (#P6040L; New England BioLabs, Ipswich, MA) in vitro phosphorylation was performed on the peptide according to the manufacturer’s instructions. Control experiments were conducted in the same way only without the kinase added to the reaction solution. Next, samples were reduced with 10 mM DTT, alkylated with 5 mM IAA, and digested with modified sequencing grade trypsin 1:50 (w/w) overnight at 37°C. Digested samples were desalted on C₈ Sep-Pak SPE columns (Waters, Milford, MA) and the eluant was dried to completeness in vacuo.