Smart system

Purified recombinant CglEndoMS (1.2 nmol), CglEndoMS-ΔCter (1.2 nmol), Cglβ-clamp (2 nmol) were applied to a Superdex 200 3.2/30 column (GE Healthcare), and were eluted at a flow rate of 40 μl/min in buffer containing 25 mM HEPES, pH 7.5, 0.5 mM DTT, 0.1 mM EDTA and 0.15 M NaCl using SMART system (Amersham Pharmacia). The standard marker proteins, including thyroglobulin (MW, 670 000), γ-globulin (MW, 158 000), ovalbumin (MW, 44 000) and myoglobin (MW, 17 000), were subjected to gel filtration as a control.

The assay was performed as previously.³² (link) Pancreatic tissue cytosolic fractions were loaded onto a Superdex S200 column using the SMART system (Amersham Biosciences, Amersham, UK). The column was equilibrated in a buffer containing (mmol/L) 50 Tris/HCl (pH 7.5), 100 NaCl, 8 MgCl₂, 2 EDTA, and 1 dithiothreitol, supplemented with 10 μmol/L GDP, at a flow rate of 50 μL/min. The samples were injected, and the material eluting between 1.2 mL and 3.3 mL was collected in 42 50-μL fractions. Ten-μL aliquots of the fractions were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (on 4%–20% gel), transferred to nitrocellulose membranes, and Rab9 and RabGDIα/β proteins identified by IB. All the IBs (Figure 3 for the full, uncropped IBs) were developed together. Fractions 1–5 and 39–42 had no proteins recognized by the antibodies; therefore, these fractions are not shown in the figures.

Gel filtration chromatography was performed using the SMART system (Amersham Pharmacia). In the experiments testing the PolD–GINS and PolD–GINS–GAN complex formation, each protein and mixture were incubated for 3 min at 60°C. Aliquots (20 μl) of the protein solution was applied on a Superose 6 PC 3.2/30 column (GE Healthcare) and eluted with a buffer containing 50 mM Tris–HCl, pH 8.0 and 0.15 M NaCl. Aliquots (0.1 μl) of the applied solution and the aliquots (4 μl) of each fraction from the eluates were subjected to 10% SDS-PAGE containing WIDE RANGE Gel Preparation Buffer (Nacalai Tesque), followed by silver staining. In these experiments testing the DP1N–Gins51C interaction, each protein (120 μM) and mixture were incubated for 5 min at 60°C, and were applied on a Superdex 200 PC 3.2/30 column (GE Healthcare). The aliquots (4 μl) of each fractionated solution eluted with a buffer containing 50 mM Tris–HCl, pH 8.0 and 0.3 M NaCl were applied Tricine–SDS–10%T, 2.6%C PAGE, followed by CBB staining. The standard marker proteins, including thyroglobulin (670 000), γ-globulin (158 000), ovalbumin (44 000) and myoglobin (17 000), were also subjected to the same gel filtration as controls.