5 ml desalting column

The single cysteine residue in RepA-WH1(A31V,K132C) was labeled with Alexa Fluor 488 maleimide (Invitrogen) using thiol chemistry. Briefly, the protein (∼50 μM) was dialyzed against 20 mM sodium phosphate buffer (pH 7.0) and 100 mM Na₂SO₄. After dialysis, TCEP [Tris(2-carboxyethyl) phosphine-hydrochloride] was added to reach a concentration of 2 mM and urea to reach a concentration of 2 M and the reaction mixture was incubated for 1 h on ice. TCEP maintains the cysteine residue in a reduced form, available for derivatization. The fluorophore was prepared by dissolving 250 μg in 10 μl H₂O and was added dropwise to the protein solution in a small vial under conditions of gentle agitation. We used a 5-fold molar excess of the dye to protein at 50 μM. From that point onward, the sample was protected from light using aluminum foil. The conjugation reaction was allowed to proceed at RT for 4 h under conditions of mild agitation. After protein labeling, unconjugated Alexa Fluor 488 was separated by the use of a 5-ml desalting column (GE Healthcare). The sample was divided into aliquots, flash frozen in N₂(l), and stored at −70°C after addition of glycerol to reach a concentration of 10%. Each aliquot was thawed immediately before the experiment and used only once. The fraction of protein labeled was 20% (i.e., ∼0.2 mol of dye per mole of protein).

A 4:3:1 mixture of phosphatidylcholine, cholesterol and stearylamine (Sigma) in chloroform was prepared at a total lipid content of 2 mg and dried under N₂. The lipid film was rehydrated with 60 mM calcein (Invitrogen) dissolved in 50 mM Na₂CO₃, pH 10.0 at 40°C for 1 h. The prepared vesicles were repeatedly extruded through a 100 nm polycarbonate membrane using a mini-extruder (Avanti), and free calcein was removed by a 5 ml desalting column (GE Healthcare). The concentration of LUVs was determined by a phosphorous assay [26 (link)].

Fluorescent-labeled Ub samples were prepared by covalently conjugating a mutant Ub construct (Ser20 → Cys) to thiol-reactive fluorescein 5′-maleimide. The Ub^S20C mutant, upon purification, was buffer exchanged into 20 mM sodium phosphate (pH 7.4) containing 0.1 mM of TCEP using a 5 ml desalting column (GE Healthcare Lifesciences, Sweden). Labeling was achieved by incubating the protein with a two-fold molar excess of freshly prepared fluorescein-5′-maleimide in DMSO (Thermo Pierce Scientific LLC, U.S.A.) at 25°C for 1 h in the dark. Unreacted dye molecules were quenched with the addition of 10 mM β-mercaptoethanol and removed from the protein using the 5 ml Hitrap desalting column equilibrated with 20 mM Tris–HCl (pH 8.0). The labeled protein was concentrated and stored at −80°C in small aliquots.