Hiload 16 60 superdex 200

For analysis of the EIIA^Glc-EAL complex, a 0.5 ml reaction containing 33 μM EAL, 78 μM EIIA^Glc or both proteins were incubated at 4 °C for 30 min and subjected to gel filtration analysis using an AKTA-FPLC system (Superdex 200, HiLoad™ 16/60, 120ml, GE Healthcare), and subsequently eluted at 4°C with a flow rate of 1 ml/min in buffer containing 20 mM Tris HCl pH 7.5, 100 mM NaCl, 1mM DTT. For CsrD variants, 0.5 ml samples containing purified CsrD^ΔTM, CsrD^ΔEAL, CsrD^ΔHAMP, CsrD^ΔCoil, or CsrD^ΔGGDEF was separated on the same system. Fractions (3 ml) were collected and analyzed by SDS-PAGE and Coomassie blue staining. For EIIA^Glc-EAL analysis, the column was pre-calibrated using 5 gel filtration molecular weight markers (1, sweet potato β-amylase, 200 kDa; 2, yeast alcohol dehydrogenase, 150 kDa; 3, bovine serum albumin, 66 kDa; 4, carbonic anhydrase from bovine erythrocytes, 29 kDa; 5, horse heart cytochrome C, 12.4 kDa), and blue dextran 2000. For CsrD variants analysis, the column was calibrated using 5 molecular weight markers (1, equine spleen apoferritin, 443 kDa; 2, sweet potato β-Amylase, 200 kDa; 3, alcohol yeast dehydrogenase,150 kDa; 4, bovine serum albumin, 66 kDa; 5, carbonic anhydrase from bovine erythrocytes, 29 kDa), and blue dextran 2000. The relative molecular masses of proteins or protein complexes were calculated by logarithmic interpolation from the standards.

Using a FPLC system (Äkta, GE Healthcare), equilibrate a Superdex 200 HiLoad 16/60 size exclusion column (GE Healthcare) in column buffer (20 mM Hepes pH 7.7, 150 mM NaCl, 0.5 mM DTT). Load 1 to 2 mL of the peak fraction(s) eluted from the Ni-NTA column and develop the size exclusion column with 1.25 column volume of column buffer, collecting fractions of eluted protein. Using UV absorbance and/or analysis of fractions by SDS/PAGE gel electrophoresis, determine the peak fractions containing the purified GBP fusion protein. Add glycerol to the peak fractions to a final concentration of 5% and freeze the GBP fusion in 25-50 μl aliquots. To determine the Stokes radius of the GBP fusion, compare its elution volume to those of standards of known size (such as Bio-Rad Gel Filtration Standard #151-1901). Specifically, use the total column volume, V_C, the void volume of the column, V₀, and the elution volume, V_e, to calculate the partition coefficient, K_av, for the GBP fusion and for the standards according to the equation: $$K_{av}=\frac{V_e-V_0}{V_C-V_0}$$ The stokes radius, R_s, can then be determined from the relationship: $$R_{\mathrm{S}}\propto \sqrt{-\log K_{\mathrm{av}}}$$