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Figure 1
Flowchart of the UW-PPG protein-purification protocol. Eight SSGCID targets were purified per week utilizing two research scientists, two ÄTKAexplorer 100s and four ÄTKAprimes (GE Healthcare, Piscataway, New Jersey, USA). Following initial immobilized metal-affinity chromatography (IMAC) of the soluble lysates, the polyhistidine tag was removed from the recombinant protein using 3C protease. The cleaved protein was separated from the 3C protease, the His-tag peptide, uncleaved protein and any Ni-binding contaminants through subtractive IMAC. Size-exclusion chromatography (SEC) was then used as a final purification step and SDS–PAGE was used to determine the fractions to pool. The pooled protein was concentrated to 20–30 mg ml−1 and stored at 193 K. In our group, the procedures were carried out on the days noted in the upper right-hand corner of each box.

ISSN: 2053-230X
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