Comparison of total protein and GFP across the two-step purification process.

<p>GFP was sequentially purified by HIC and SEC, as described above, and aliquots from each step were compared side-by-side using Coomassie stain (<i>A</i>), western blot (<i>B</i>), and ELISA (<i>C</i>) to assess changes in GFP purity and concentration. GFP-containing bacterial lysate (<i>Lysate</i>) was loaded onto the phenyl-34 column and fractioned using HIC. The contiguous HIC fractions containing high levels of retained GFP were collected (<i>HIC Pooled</i>) and then concentrated (<i>HIC Conc.</i>). The concentrated GFP-containing HIC eluate was further separated from contaminant proteins by SEC, and SEC fractions containing retained GFP were collected (<i>SEC Pooled</i>) and then concentrated (<i>SEC Conc.</i>) to yield the final, purified GFP product. An equal volume of <i>HIC Pooled</i>, <i>HIC Conc.</i>, <i>SEC Pooled</i>, and <i>SEC Conc.</i> was electrophoresed in in order to permit a direct visual comparison of protein concentration across the purification procedure. The volume of <i>Lysate</i> electrophoresed was half the volume of the other samples in order to prevent overloading of the SDS-PAGE gel. The western blot shown in <i>B</i> was probed using anti-GFP antibody, and the full-length membrane is displayed in order to demonstrate the high degree of antibody specificity. The ELISA in <i>C</i> quantifies the concentration of GFP in each step of the purification. Samples were assayed in triplicate and bars represent the average concentration ± SEM.