Strep tactin superflow agarose

Phosphorylated or non-phosphorylated wild-type or mutant Sli15-2xStrep-HA-6xHis/Ipl1 was immobilized on Strep-Tactin Superflow agarose (Qiagen). For prephosphorylation, Sli15/Ipl1 was incubated at 30°C for 30 min in the presence of 3 mM MgCl₂ and 3 mM ATP. Samples for non-phosphorylated Sli15/Ipl1 were treated the same way, but instead of 3 mM ATP the non-hydrolysable analog AMP-PNP (Santa Cruz Biotechnology) was applied. To remove basal phosphorylation, Sli15/Ipl1 was treated with lambda phosphatase (New England Biolabs) at 30°C for 30 min. Subsequently, non-phosphorylated as well as phosphorylated or dephosphorylated Sli15/Ipl1 complexes were washed three times with binding buffer [50 mM NaH₂PO₄(pH 8), 120 mM NaCl, 5% glycerol].
Testing of binding between Ame1/Okp1, Ctf19/Mcm21 and Sli15/Ipl1 was performed in binding buffer at 4°C, 1000 rpm for 1 hr in a thermomixer (Eppendorf). Unbound proteins were removed by washing three times with binding buffer. The complexes were either eluted with 8 mM biotin in 50 mM NaH₂PO₄(pH 8), 500 mM NaCl, 5% glycerol or by boiling in 2x SDS loading buffer.
To quantify the ratios of bound proteins to the bait protein SDS page band intensities were analyzed by using the Fiji software (Schindelin et al., 2012 (link)).

Expression constructs for 6xHis-Chl4/Iml3, 6xHis-Cnn1/Wip1-1xFlag, 6xHis-Nkp1/Nkp2 and Mhf1/Mhf2-1xStrep were created by amplification of genomic DNA and cloned into pETDuet-1 vector (Novagen). Expression was performed in BL21 (DE3) cells (New England Biolabs). Cells were grown at 37°C until OD₆₀₀ 0.6, followed by induction with 0.5 mM IPTG for Chl4/Iml3 or 0.2 mM IPTG for all other protein expressions. Protein expression was induced overnight at 18°C, or for 3 hr at 23°C, respectively.
Cells were lysed using a French Press in lysis buffer [50 mM Hepes (pH 7.5), 400 mM NaCl, 3% glycerol, 0.01% Tween20 and cOmplete ULTRA EDTA-free Protease Inhibitor Cocktail (Roche)]. 6xHis-tagged proteins were purified using Ni-NTA agarose (Qiagen), whereby 30 mM imidazole were added to the lysis buffer in the washing step, followed by protein elution in 50 mM Hepes pH 7.5, 150 mM NaCl, 300 mM imidazole, and 5% glycerol. Strep-tag purification was performed using Strep-Tactin Superflow agarose (Qiagen) and eluted in a buffer containing 50 mM Hepes (pH 7.5), 150 mM NaCl, 8 mM biotin and 5% glycerol.
Buffer exchange into a buffer containing 50 mM Hepes (pH 7.5), 150 mM NaCl and 5% glycerol was performed using a Superdex 200 HiLoad 16/60 column (GE Healthcare) for Chl4/Iml3 and Cnn1/Wip1 or using a PD10 desalting column (GE Healthcare) for Nkp1/2 and Mhf1/2 protein complexes.