Standard proteins

The purified proteins were exchanged into a low-salt system (20 mM NaH₂PO₄, 50 mM NaCl, pH 7.8) and run as in the purification step. Each run with the proteins was preceded by a run with a mixture of standard proteins (GE Healthcare) [aprotinin (6,500 Da), ribonuclease (13,700 Da), ovalbumin (44,000 Da), conalbumin (75,000 Da), aldolase (158,000 Da), ferritin (440,000 Da)] to generate a K_av vs. logMW curve, where K_av = (V_e − V_o)/(V_c − V_o). Proteins were eluted at a flowrate of 0.8 mL/min, and the elution volumes were determined using the Unicorn software integration function. The resulting standard curve was used to estimate the molecular weight for our proteins.

MWs were estimated by gel filtration chromatography on an FPLC Akta Purifier 10 (GE Healthcare Bio-sciences AB, Uppsala, Sweden) equipped with a separation range of 1–300-kDa column Superose 12 10/300 GL. The standard proteins (GE Healthcare, Chalfont Saint Giles, UK) were used to calibrate the column: blue dextran (2000 kDa), aldolase (158 kDa), conalbumin (75 kDa), bovine serum albumin (67 kDa), ribonuclease A (13.7 kDa) and bacitracin (1.423 kDa). A calibration line was made using the logarithms of the MWs of these standard proteins and their elution volumes. For the elution, 50 mL of 0.05-M sodium phosphate buffer, 0.5-M sodium chloride and 0.02% (w/v) sodium azide adjusted at pH 7.5 with a flow of 1 mL/min was used. The concentration and injected volume of the samples were 30 mg/mL of protein and 500 μL, respectively. Protein elution was recorded by measuring its absorbance at 280 nm.