Figure 4

Partial purification of P2RX4 proteins by Accudenz DGU. (A) Floatation of GFP-P2RX4/lipid complex by Accudenz DGU. GFP and GFP-P2RX4 were synthesized using the wheat cell-free system supplemented with liposomes. After the synthesis reaction, the reaction mixture was subjected to Accudenz DGU. Fluorescence images were visualized using a transilluminator. (B) Yield of synthesized protein in each fraction. After DGU, fractions were collected from the top of the tube and the fluorescence intensity of each fraction was measured with a spectrofluorometer. (C) Effect of centrifugation time on floatation of lipid/MP complex. Synthesized GFP-fusion MPs were subjected to Accudenz DGU at varying centrifugation times. After DGU, fractions were collected from the top of the tube and the fluorescence intensity of each fraction was measured using a spectrofluorometer. (D) The effect of centrifugation time on the floatation of liposomes containing different MPs. GFP-P2RX4, -P2RX1, and -SLC6A18 fusion proteins were used in this experiment. Fraction No. one was treated as the liposome fraction and its fluorescence intensity in each condition was plotted. (E) Partial purification of P2RX4 proteins. P2RX4 was synthesized using the wheat cell-free system supplemented with liposomes. After the synthesis reaction, the reaction mixture was concentrated and subjected to Accudenz DGU. The synthesis mixture and the top lipid/MP complex fraction after DGU were applied to SDS-PAGE and stained by CBB.