Fig. 2.
Automated Purification of hPCNA. (A) Typical chromatogram for the automated 4-step purification of hPCNA using ÄKTAXpress™. The pre-packed columns used are illustrated above the corresponding section of the chromatogram; IEX – ion-exchange, DS – desalt, AF – affinity, GF – gel-filtration. Solid black; A280 nm in mAU (left axis). Solid red; elution gradient in % Buffer-E or Buffer-G (right axis), IEX and AF step, respectively. The buffer pairs used are indicated above the appropriate portion of the chromatogram. The inset details the region of the gel-filtration column elution from which fractions were collected. Indicated fractions A7–C11 were pooled. (B) SDS–polyacrylamide gel (4–20% gradient) illustrating the final purity levels of hPCNA purified by both manual (⩾93%) and automated (⩾87%) protocols (determined by gel densitometry). The final purity from 2 independent traditional and 2 independent automatic runs are shown, illustrating the excellent reproducibility of both methods. Five μg total protein was loaded in each lane. SCE, soluble cell extract; R1, run 1; R2, run 2. Molecular weight markers are shown to the right of the gel. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)