Superdex 75 pc 3.2 30 column

Analytical size exclusion chromatography was performed on an AKTA Micro System (GE Life Sciences) using a Superdex 75 PC 3.2/30 column equilibrated in: 20 mM Tris pH 7.4, 150 mM NaCl, 2 mM DTT. In total 25 μl of 80 μM of each sample was loaded onto the column. Complexes were mixed at an equimolar ratio and incubated at room temperature for 10 min prior to loading onto the column. Fractions containing proteins were mixed with SDS sample loading buffer and subjected to SDS-PAGE analysis.

SEC was performed at a flow rate of 0.1 ml/min using a Superdex 75 PC 3.2/30 column (GE Healthcare, UK) equilibrated with the appropriate mobile phase. Eluted peaks were detected at 406 nm.
When a buffered 20% ACN mobile phase was used, 20 mM of AMB-PEG conjugate formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in ACN-acetic-acid-water (20:4.3:75.7 v/v/v). Precipitates were removed by centrifugation at 14000 g. Along with the peak fractions collected earlier from RPC, 50 μL of these samples were then analyzed by SEC using a mobile phase composed of the same ACN-acetic acid-water mixture. All samples were analyzed neat except for unconjugated AMB that was diluted tenfold prior to analysis.
For SEC performed using PBS-EDTA as the mobile phase, 3 μL of 2 mM AMB-PEG conjugate formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analyzed. All samples dispersed in PBS-EDTA were 0.22 μm-filtered (Millipore) prior to column injection.