Purification of Mitochondrial Proteins HSP60 and ATP Synthase from Ascidian Eggs: Implications for Antibody Specificity
Figure 5
Purification of p63 using successive DEAE ion exchange columns and IEF-PAGE.
In A, C and D, pairs of identical gels show distribution of p63 (western blot with PMF-C13, lower gels) compared to total protein (upper gels). (A) Total Phallusia egg extract prepared at low ionic strength was passed through an ion exchange column, eluted with a step salt gradient and representative fractions were analyzed; p63 (arrowhead) elutes at 100–200 mMNaCl but is not well separated from the majority of proteins. (B) Schematic of biochemical approach used to enrich p63 before 2D-electrophoresis. (C) Scale-up and sequential ion exchange columns. Column 1: The majority of proteins are retained on the overloaded column (“elute peak”) but p63 (arrowhead) passes through. The flow through (FT) from the first column (left) was loaded onto a second column as indicated by the curved arrow. Column 2: p63 elutes at 200 mM salt as in A. A fraction enriched for p63 (black rectangle) was loaded onto the duplicate 2D gels shown in D: the p63 spot (arrow) is abundant and well separated from other proteins.