Figure 4
Purification, verification, and transduction of exogenous p16INK4a fusion protein. a. Successful expression and purification of the p16INK4a fusion protein was confirmed by 12% SDS-PAGE analysis. The bacterial sample before IPTG induction showed almost no protein expression (lane 1). After IPTG induction and centrifugation, p16INK4a fusion protein was abundant in the clear supernatant (lane 3) (indicated by the arrow) and absent from the bacterial precipitate (lane 2). The supernatant was loaded onto a Ni2+-affinity chromatography column, which binds the His-p16INK4a fusion protein. Nonspecifically bound proteins were removed with washing buffer; the flow-through liquid can be seen in lane 4. Elution buffer with different concentrations of imidazole was used to elute the p16INK4a fusion protein: 20 mM (lane 5), 50 mM nt (lane 6), 100 mM (lane 7) and 200 mM (lane 8) were. The fractions were assessed by SDS-PAGE and the sample corresponding to the 100 mM imidazole eluent (lane 7) was found to contain p16INK4a fusion protein of high purity (arrow). b. The purified protein was verified by Western-blot analysis using the specific p16INK4a antibody. c. Immunocytochemical assay to assess transduction efficiency. All nuclei of A549 cells stained with Hoechst fluorescent and the exogenous p16INK4a protein was detected in about 50% of cells, as shown by the FITC signal. As shown in the figure, the transduction efficiency was about 50%.