0.2 μm syringe filter

The reaction mixture was adjusted to pH 7.0 using 1 N HCl. The Fc-containing protein was separated from SrtA_Δ1–59 and unreacted PLP peptide using protein G affinity chromatography. The column was first equilibrated using TBS, pH 7.0. Then the reaction mixture was loaded onto the Protein G column, and the captured Fc protein was washed with 500 mL of 50 mM Tris and 500 mM NaCl, pH 7.0. Then the Fc-containing protein (unreacted LABL-Fc-ST and product LABL-Fc-ST-PLP) was eluted using 0.1 M glycine-HCl buffer pH 2.7 and immediately dialyzed against TBS, pH 7.5. The protein was then passed through a Detoxi-Gel endotoxin removal column (Thermo Scientific) to remove endotoxin contaminants that may have been introduced during the expression and/or purification steps.^{52 (link)} The flow through from the endotoxin removal column was concentrated using an EMD Millipore 10 kDa MWCO centrifugal concentrator (USA). Concentration was stopped when the volume of the protein solution was 2 mL because precipitation was observed. The concentrated protein was dialyzed against 4 L of 1× PBS pH 7.4 with one buffer change at 4°C overnight and then filtered through an EMD Millipore 0.2 μm syringe filter. The concentration of the end product was determined to be 168 μM (9.6 mg/mL, 19 mg total) by optical density of the solution at 280 nm (MW = 57128.3 Da, ε₂₈₀ = 82570 M⁻¹cm⁻¹).^{51 (link)}