The column was washed with loading buffer until the background ultraviolet absorption was negligible. Elution of protein was carried out with the loading buffer containing 1 M NaCl, and protein was concentrated and injected onto a Superose 6 Increase 10/30 GL gel filtration column preequilibrated with 20 mM tris-Cl (pH 8.0) and 250 to 300 mM NaCl. Superose 6 Increase 10/300 GL is a versatile, prepacked column for high-resolution size exclusion chromatography in small-scale ( Increased resolution compared with Superose 6, for higher purity and improved analysis results. Reduced runtime compared with Superose 6, for rapid results. Versatile use Fractionation range of globular proteins and peptides of various molecular weights on Superdex™ 30 Increase 10/300 GL, Superdex 75 Increase 10/300 GL, Superdex 200 Increase 10/300 GL, and Superose™ 6 Increase 10/300 GL prepacked chromatography columns. Note that the whole fractionation range of Superose 6 Increase is not covered in this diagram. well-establishedabilityofSECresins,suchasSuperdex™,Superose™, Sephacryl™,Sephadex™andSepharose™toseparatemolecules accordingtosize.Prepackedcolumnsareavailable Superdex75Increase3.2/300 3×103to7×104 2.4 10 Superdex200Increase10/300 1×104to6×105 24 100 GL Superdex200Increase5/150 1×104to6×105 3 12.5 The undigested IgGs were removed by SEC using a Superose 6 increase 10/300 column (GE Healthcare Biosciences). The purified Fabs were concentrated and assessed by SDS-polyacrylamide gel electrophoresis for purity. Iterative rounds of Rosetta relaxed refinement and manual Coot refinement were applied to generate the final models (29, 51, 52). Use the ENrich SEC 650 10 x 300 column for completing size exclusion separations in less than 30 minutes at fast flow rates (≥1 ml/min). For biomolecules in the 5-650 kDa molecular weight range. The ENrich SEC columns run at low backpressure, allowing for more flow rate flexibility, both in terms of sample viscosities and temperature Size Fractionation by Superose 6 Gel Filtration. 1. Superose 6 analytical grade column: HR 10/30 with a total bed volume of 24 mL. Preparative grade column: XK 50/600 with a bed height of 30.3 cm, total bed volume of 589 mL (both from Amersham Biosciences, UK). Range of separation of 5000 to 5,000,000 Daltons. For gel filtration analysis, rat brain tissue from Sprague Dawley rats (embryonic day 18) was extracted in lysis buffer (0.25 M sucrose, 50 mM Tris-HCl, pH 7.4, 50 mM NaCl, and 0.5% Triton supplemented with EDTA-free protease inhibitor tablets from Roche [11873580001]) by manual dicing using a razor blade on a glass Petri dish followed by 2.6 ml/min for 26/600) Sample volume 0.5% to 4% of the CV (0.6 to 4.8 ml for 16/600 or 1.6 to 12.8 ml for 26/600) Note: Sample volume is critical for the separation. Sample preparation Dissolve the sample in runn ing buffer, filter through 0.22 μm filter or centrifuge at 10 000 x g for 10 min Buffer 0.05 M NaPO 4, 0.15 M NaCl, pH 7.2 or select Start a slow flow rate of buffer (0.5 ml/min or less) through the column. 2. Remove the lower end tubing and fitting from the column. Firmly hold the bottom of the column over a sink or container. 3. Loosen the lock nut by turning it clockwise. 4. Raise the adaptor a few millimeters by slowly by turning the adjusting nut counterclockwise. 5. (1) Connect a syringe or pump to the storage/shipping device and fill with 20% ethanol over the mark on the tube. Remove the syringe or connection to the pump.