Whatman chromatography paper grade 1chr

UV spectra were recorded on a Shimadzu UV-1601PC spectrophotometer in spectroscopic grade MeOH. IR spectra were recorded on a Shimadzu IR-460 spectrometer in pressed KBr disks. NMR spectra were recorded on Bruker AMX 400, operating at 400 MHz for proton and 100 MHz for carbon in spectroscopic grade CDCl₃, DMSO-d6, and CD₃OD. EI-MS (70 eV) were recorded on Agilent 7890A/5975C. TLC was carried out on precoated silica gel 60 F 254 aluminum sheets (Merck, Rahway, NJ, USA). Whatman chromatography paper (Grade 1 CHR) was obtained from GE Healthcare Life Sciences (Shanghai, China) and used with further adjustment of size. All chromatographic solvents were of reagent grade (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). For column chromatography, normal phase silica gel 60 (70–230 mesh, Merck), Sephadex LH-20 (25–100 μm, Sigma-Aldrich, St. Louis, MO, USA), and polyamide (particle size 50–160 μm, Sigma-Aldrich) were used.

Cellulose-bound peptide arrays were prepared using standard Fmoc solid phase peptide synthesis on a MultiPep-RSi-Spotter (INTAVIS, Köln, Germany) according to the SPOT synthesis method provided by the manufacturer, as previously described (Picaud and Filippakopoulos 2015 (link)). In brief, twenty-one-residue-long peptides based on MITF residues 314 to 333 were synthesized on amino-functionalized cellulose membranes (Whatman Chromatography paper Grade 1CHR, GE Healthcare Life Sciences #3001–878) and the presence of SPOTed peptides was confirmed by ultraviolet light (UV, λ = 280 nM). The wild-type sequence was mutated or modified where indicated. The membrane was quickly washed in EtOH 100% then equilibrated overnight in Kinase Buffer (HEPES 50 mM pH7.5, NaCl 200 mM, MgCl2 10 mM, KCl 10mM). For in vitro kinase assays, the membrane was then incubated in Kinase Buffer containing 500 ng of kinase for 1 h at 30 °C. After washing, the membranes were analysed by western blot. The wildtype sequence was mutated or modified where indicated. The membrane was briefly incubated in EtOH 100% followed by an overnight incubation in the Kinase Buffer (HEPES 50mM ph7.5, NaCl 200 mM, MgCl₂ 10 mM, KCl 10mM). For the in vitro kinase assays, the membrane is then incubated in the Kinase Buffer containing 500 ng of the kinase for 1 h at 30°C. After washing, the membrane is analysed by western blot.