Superdex 75 16 26 gl

Human Dok1 PTB domain (Q154 – G256; Swiss-Prot Q99704, number as Q1-G103) was sub-cloned into the pET14b vector with an N-terminal six His-tag. Escherichia coli Rosetta cells were transformed with the expression plasmid and grown at 37 °C either in LB or M9 medium containing [¹⁵N] ammonium chloride (Cambridge Isotope Laboratories). Expression of recombinant protein was induced by adding 1 mM IPTG to cells (OD₆₀₀ 0.6–0.7) followed by incubation for 6–12 h at 16 °C. Cells were centrifuged and re-suspended in Buffer A (20 mM Tris-HCl buffer, pH 8.0) followed by affinity purification on a Nickel-NTA column (Qiagen). His-tagged Dok1 PTB domain was eluted in Buffer A containing 500 mM imidazole and dialyzed against Buffer B (50 mM sodium phosphate buffer (pH 6.0) containing 100 mM NaCl and 2 mM DTT). Protein samples were further purified by gel filtration in Buffer B on a Hiload Superdex 75 16/26 GL preparative column that was connected to an AKTA FPLC UPC-900 system (GE Healthcare UK Ltd., England). Proteins were eluted at a flow rate of 0.5 ml/min and monitored by absorbance at λ = 276 nm.

His tagged human Dok1 PTB domain (Q154 – G256; Swiss-Prot Q99704) was sub cloned into the pET14b vector and over expressed into Escherichia coli Rosetta cells, grown at 37°C either in LB or M9 medium containing [¹⁵N] ammonium chloride and [¹³C] glucose (Cambridge Isotope Laboratories). Cells were induced with 1 mM IPTG at an OD₆₀₀ of 0.6–0.7 and incubated for 6–12 hours at 16°C for protein production. Cells were centrifuged and resuspended in 20 mM Tris-HCl buffer, pH 8.0. Cell solutions were lysed by sonication and the supernatant was applied to Nickel-NTA column for His tag-based affinity purification (Qiagen). The weakly bound proteins were removed from the column by extensive washing with appropriate buffers. Target protein was eluted with buffer containing 500 mM imidazole. Further, purified protein was extensively dialyzed against 50 mM sodium phosphate buffer, containing 100 mM NaCl and 2 mM DTT at pH 6.0. Samples of Dok1 PTB domain were loaded onto a Hiload Superdex 75 16/26 GL preparative column with the AKTA FPLC UPC-900 system (GE Healthcare UK Ltd., England). FPLC column was equilibrated with 50 mM sodium phosphate buffer containing 100 mM sodium chloride and 2 mM DTT, pH 6.0. Protein samples were eluted at a flow rate of 0.5 ml/min and detected spectrophotometrically at 276 nm. The amino acid sequence of Dok1 PTB domain was numbered as Q1- G103.