Figure 2
From: Cloning, expression and purification of functionally active human angiopoietin-like protein 2
Purification of recombinant human Angptl2-GST from conditioned media . After loading the GSTrap column with 2–2.5 liters of conditioned media, the column was washed with TBSE and then eluted with 10 mM glutathione in TBSE. The fractions obtained during the elution of Angptl2-GST from the GSTrap column were resolved on SDS-PAGE to assess the specificity of the purification. A) Following SDS-PAGE, proteins were detected by staining with Coomassie Brilliant Blue. Lane 1 of each gel contains molecular mass markers. Lanes 2–10 contain the indicated fraction number (5 μl each). B) Immunoblotting of the GSTrap elution profile. The samples loaded were 0.1 μl of the same fractions in panel A, loaded on a separate set of 10% acrylamide gels and Immunoblotted as described in Methods. The membranes were probed using an antibody specific for human Angptl2 antibody. C) Quantification of purified Angptl2-GST. An aliquot (2 μl) of purified Angptl2-GST was resolved on SDS-PAGE (10% acrylamide) along with a standard curve of BSA (0.1, 0.2, 0.3, 0.5, 1 and 2 μg). The gels were stained with Coomassie Brilliant Blue R-250, digitized using a two-dimensional gel scanner, and band intensities for BSA and Angptl2-GST determined using Quantity One software and the quantity of Angptl2-GST determined from a linear regression analysis of a plot of band intensity versus micrograms of BSA loaded using GraphPad Prism software.