Protease inhibitor cocktail edta free

Tubulin was isolated from pig brain by two cycles of microtubules polymerization/depolymerization in high molarity pipes buffer as described in Castoldi and Popov (2003) (link).
MBP-Kip2-(1-560)WT-RFP and MBP-Kip2-(1-560)P1⁻-RFP constructs were cloned in pET(24)d plasmid and expressed in E. coli (Rosetta strain). Bacteria were grown in 2 YT medium at 37°C until culture reached OD 0.6 and then the recombinant protein expression was triggered by the addition of 200 µM 1-thio-β-D-galactopyranoside for 16 h at 16°C. Cells were harvested and resuspended in purification buffer for Kip2-RFP (2XT buffer: 40 mM Tris, 400 mM KCl, 2 mM MgCl2, 1 mM ATP, pH 7.4) or for mCherry-Bik1 (2XH buffer: 40 mM Hepes, 400 mM KCl, 2 mM MgCl2, 1 mM ATP, pH 7.4) with 5 mM β-mercaptoethanol and protease inhibitor (SigmaFAST, protease inhibitor Cocktail EDTA-Free, S8830) and lysed by sonication, and the protein purified using Amylose resin (E8021; NEB) and eluted with 10 mM maltose. The proteins were aliquoted, flash frozen in liquid nitrogen, and stored at −80°C.

We lysed batches of ~9×10⁹ cells by completely resuspending frozen cell pellets in 25 ml ice-cold urea-based cell hybridization buffer (25 mM Tris pH 7.5, 500 mM LiCl, 0.25% dodecyl maltoside, 0.2% sodium dodecyl sulphate, 0.1% sodium deoxycholate, EDTA 5 mM, TCEP 2.5 mM, and 4 M urea). Next, the cell sample was passed ~10 times through a 27-gauge needle attached to a 25 ml syringe in order to disrupt the pellet and shear genomic DNA. At this point lysates were kept at −80°C or incubated at 65°C for 10 min before clearing by centrifugation for 10 min at 10,000 × g.
For the first purification, frozen cell pellets were resuspended in 8 ml ice-cold detergent-based cell lysis buffer (10 mM Tris pH 7.5, 500 mM LiCl, 0.5% dodecyl maltoside, 0.2% sodium dodecyl sulphate, 0.1% sodium deoxycholate, 1× Protease Inhibitor Cocktail EDTA-free, and 900 U of Murine RNase Inhibitor) (New England Biolabs), and cell sample passed 10–15 times through a 27-gauge needle attached to a 25 ml syringe in order to disrupt the pellet and shear genomic DNA. The samples were then treated for 10 min at 37°C adding 1× DNAse salt solution (2.5 mM MgCl₂, 0.5 mM CaCl₂), and 900 U of DNase I [Roche] to digest DNA. Samples were returned to ice and the reaction was immediately terminated by the addition of 16 ml 1.5× cold hybridization buffer to stop reaction.