Sp fast flow

Oligonucleotides (25 nmol scale, standard desalting) were from Integrated DNA Technology (San Diego, CA, USA). Commercial lysostaphin (ssLys; short for Staphylococcus simulans lysostaphin) and SYPRO Orange 5000× Protein Stain were from Sigma (St Louis, MO, USA). MicroAmp^® Fast Optical 0.1 ml 96-Well Plates and MicroAmp^® Optical Adhesive Film were from Applied Biosystems (Bedford, MA, USA). Restriction enzymes and PCR reagents were purchased from New England BioLabs (Ipswich, MA, USA). Plasmid purification kits and Ni-NTA resin were from Qiagen (Valencia, CA, USA). Growth medium was from Becton Dickinson (Franklin Lakes, NJ, USA). PCR cleanup and gel extraction kits were from Zymo Research (Irvine, CA, USA). SP Fast Flow was from GE Healthcare Life Sciences (Little Chalfont, Buckinghamshire, UK). SYTOX® Green nucleic acid stain was purchased from Life Technologies (Carlsbad, CA). MBEC^™ Biofilm Inoculator 96-well plates were purchased from Innovotech (Edmonton, AB, Canada). Unless noted, all other chemicals and reagents were from Fisher Scientific (Pittsburgh, PA, USA).

E. coli BL21(DE3) containing pET28 UbcH5c was grown at 37 °C and induced with 1.0mM IPTG for 24 hours at 16 °C. Cell pellets were resuspended in 30mM MES pH 6.0 and disrupted by sonication (3 cycles of 30 second pulses). The extracts were cleared by centrifugation and applied to SP Fast Flow (GE). UbcH5 was eluted with a 0–0.5M NaCl gradient in 30mM MES pH 6.0 with 2mM DTT and 0.5mM PMSF. Fractions containing UbcH5 were purified by size-exclusion chromatography in 25mM sodium phosphate pH 7.0, 2mM DTT.
E. coli BL21(DE3) containing pGEX6p-1 CHIP was grown at 37 °C and induced with 1.0mM IPTG for 24 hours at 16 °C. Cell pellets were resuspended in NETN buffer, clarified, and applied to glutathione beads. CHIP was cleaved from glutathione beads with H3c protease and purified by size-exclusion chromatography in 50mM Tris, 50mM KCl, pH 7.5.
E. coli BL21(DE3) containing pMCSG7 Hsp72 was grown at 37 °C and induced with 1.0mM IPTG for 24 hours at 16 °C. Cell pellets were resuspended in 50mM Tris pH 7.5, 1M NaCl, 20mM imidazole. Cell pellets were disrupted by sonication (3 cycles of 30 second pulses). The extracts were cleared by centrifugation and applied to nickel agarose (Gold Biotechnology) before elution with a 0–0.5M Imidazole gradient. Fractions containing Hsp72 were then purified by size-exclusion chromatography in 50mM Tris, 50mM KCl, pH 7.5.