Tandem Affinity Purification of TLK Proteins

Tandem affinity purification (TAP) was carried out as described previously (43 (link)). Briefly, S-protein-2xFLAG-Streptavidin binding peptide (SFB)-tagged TLK1, SFB-TLK1 1–240, SFB-TLK1 D607A, SFB-TLK1 133–208, SFB-TLK1 133–208 Y149A F150A, or SFB-TLK2 were transfected into HEK293T cells. Cells were harvested 24 hours after transfection using NETN buffer (150 mM NaCl, 0.5 mM EDTA, 20 mM Tris-HCl pH 8.0, 0.5% NP-40) supplemented with 2 μg/mL aprotinin (Thermo, AAJ60237MB) and 5 μg/mL pepstatin A (Thermo, PI78432) at 4°C for 20 minutes. Lysates were centrifuged at 9,000 × g, 4°C for 20 minutes to yield supernatant as soluble fraction. The pellet was washed 1x with PBS and centrifuged at 9,000 × g, 4°C for 5 minutes and lysed with chromatin extraction buffer (NETN buffer, 10 mM MgCl₂, and Turbonuclease, Accelagen, N0103M) at 4°C for 1 hour followed by centrifugation at 9,000 × g, 4°C for 20 minutes to obtain chromatin fraction. Both soluble and chromatin fractions were incubated with streptavidin sepharose (200 μl) (GE Healthcare, GE17–5113-01) at 4°C for 1 hour followed by washing with NETN buffer three times. The protein complexes were eluted with 2 mg/mL biotin at 4°C for 1 hour. The eluents were then incubated with S-protein agarose (EMD Millipore, 69704–3) overnight at 4°C, washed three times with NETN buffer, and eluted in 1x Laemmli buffer.