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An affinity resin-based pull-down method is convenient for the purification of biochemical materials. However, its use is difficult for the isolation of a molecular complex fully loaded with multiple components from a reaction mixture containing the starting materials and intermediate products. To overcome this problem, we have developed a new purification procedure that depends on sequential elimination of the residues. In practice, two affinity resins were used for purifying a triangular-shaped RNP (RNA-protein complex) consisting of three ribosomal proteins (L7Ae) bound to an RNA scaffold. First, a resin with immobilized L7Ae protein captured the incomplete RNP complexes and the free RNA scaffold. Next, another resin with an immobilized chemically modified RNA of a derivative of Box C/D motif, the binding partner of L7Ae, was used to capture free protein. The complete triangular RNP was successfully purified from the mixture by these two steps. Obviously, the purified triangular RNP displaying three protein-binding peptides exhibited an improved performance when compared with the unrefined product. Conceptually, this purification procedure should be applicable for the purification of a variety of complexes consisting of multiple components other than RNP.

Purpose: To identify sectors of the optical coherence tomography (OCT) macular map that could be used to effectively assess structural progression in patients with normal-tension glaucoma (NTG)., Methods: This study examined 117 eyes of 117 NTG patients to establish axonal tract-dependent macular sectors, and also examined a separate group of 122 eyes of 81 NTG patients to evaluate the ability of these sectors to reveal glaucoma progression. Longitudinal data, including macular maps from at least 5 OCT examinations performed over at least 2 years, was available for all patients in this group. Circumpapillary retinal nerve fiber layer thickness (cpRNFLT), temporal clockwise sector scans (from 7 to 11 o'clock), macular retinal nerve fiber layer thickness (mRNFLT), and macular ganglion cell layer plus inner plexiform layer thickness (mGCIPLT) were measured with spectral-domain OCT (3D OCT-2000, TOPCON). The axonal tract-dependent macular sectors were identified by calculating Spearman's rank correlation coefficient for each point on a grid overlaid on the macular map and cpRNFLT in each clockwise scan sector. Trend and event analyses for the slope of progression in each sector and macular map were performed. Visual field progression in the macula was defined by the presence of more than 2 progressive test points in the 16 central test points of the Humphrey field analyzer SITA standard 24-2 program, evaluated with Progressor software., Results: The slope of progression in the entire macular area was -0.22 ± 0.58 μm/year for mRNFLT and -0.35 ± 0.52 μm/year for mGCIPLT. The fastest-progressing mRNFLT sector (-1.00 ± 0.84 μm/year, p < 0.001) and mGCIPLT sector (-1.16 ± 0.63 μm/year, p < 0.001) progressed significantly faster than the overall macula. Classifying patients according to visual field progression showed that baseline mRNFLT in the inferior hemifield, 7 and 8 o'clock sectors, as well as baseline mGCIPLT in the overall macular map, inferior hemifield, and 8 o'clock sector, were significantly lower in progressors (22 eyes) than non-progressors (100 eyes). There were significant differences in mRNFLT slope in 8 o'clock sector and in the fastest progressing sector in progressors and non-progressors, but mGCIPLT did not differ, even in the fastest-progressing sector. Event analysis showed that progression occurred most frequently in inferior mRNFLT and superior mGCIPLT in this study., Conclusion: Axonal tract-dependent OCT macular sectors could effectively reveal structural change in patients with NTG. Furthermore, mRNFLT slope was consistent with visual field progression. This method promises to open new avenues for the OCT-based evaluation of glaucoma progression.