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Figure 4 | Biological Procedures Online

Figure 4

From: Increasing the library size in cDNA display by optimizing purification procedures

Figure 4

Effect of His-tag-binding buffer in RNase T1 digestion and His-tag purification to recover mRNA/cDNA-protein fusion molecules from SA-beads. (A) cDNA-protein fusion molecules were synthesized and released from the SA-beads by RNase T1 treatment in RT reaction mixture or His-tag-binding buffer (20 mM Sodium phosphate, pH 7.4, 500 mM NaCl, 5 mM imidazole, 0.05% Tween-20). cDNA-protein fusions in the each sample were purified with 20 μL of Ni-NTA beads (His Mag Sepharose Ni, GE Healthcare Bio-Sciences, Piscataway, NJ, USA) according to the attached instruction. Inputted mRNA-linker conjugates (lane I), translation mixture (lane T), each supernatant (Sup.) of Ni-NTA beads and eluate were analyzed by 4% stacking–6% separating SDS-PAGE containing 8 M urea. (B) Formation efficiencies of mRNA/cDNA-protein fusion molecules from mRNA-linker-conjugates were estimated by comparing the band intensities between the purified cDNA-protein fusion molecule of each lane and that of the mRNA-linker conjugate (lane I). Results are the mean of three independent experiments performed in duplicate. Error bars = standard deviation.

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