IP of 3×FLAG-tagged Kss1 and mutants
This protocol is extracted from research article:
Engineering allosteric regulation in protein kinases
Sci Signal, Nov 6, 2018; DOI: 10.1126/scisignal.aar3250

Cultures (2 × 250 ml) of each strain were grown to OD600 = 0.8 at 30°C with shaking, one in SDC and the other in SDC + 20 nM estradiol. The SDC culture was left untreated, whereas the SDC + estradiol culture was treated with 1 μM αF for 30 min. Samples were collected by filtration, and filters were snap frozen in liquid N2 and stored at −80°C. Cells were lysed frozen on the filters in a coffee grinder with dry ice. After the dry ice was evaporated, lysate was resuspended in 1 ml of IP buffer [50 mM Hepes (pH 7.5), 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% deoxycholate, and complete protease inhibitors], transferred to a 1.5-ml tube, and spun to remove cell debris. Clarified lysate was transferred to a fresh tube, and serial IP was performed. First, 25 μl of anti-FLAG magnetic beads (50% slurry, Sigma) was added, and the mixture was incubated for 2 hours at 4°C on a rotator. Beads were separated with a magnet, and the supernatant was removed. Beads were washed five times with 1 ml of IP buffer, and bound material was eluted twice with 25 μl of 3×FLAG peptide (1 mg/ml; Sigma) in IP buffer by incubating at room temperature for 10 min. Beads were separated with a magnet, and the two eluates were pooled in a fresh tube. Ten microliters of eluate was analyzed by Western blotting.

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