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. 2018 Jul 30;115(33):E7824–E7833. doi: 10.1073/pnas.1800609115

Fig. 2.

Fig. 2.

Identification of the Ycf48 protein in YFP-tagged PSI complexes isolated by a single step GFP-Trap affinity pulldown. (A) GFP-Trap preparations pulled down from the strain expressing PsaF-YFP (PSI-YFP) and from the WT strain were eluted by hot SDS and analyzed by 1D SDS/PAGE. The gels were stained by SYPRO Orange; blotted to a PVDF membrane; and Ycf48, PsaD, and D1 were consecutively detected using specific antibodies (segment of the blot with the specific antibody signal is shown). A total of 2 μg of chlorophyll was loaded for WT and ycf48 membranes; 15 μL of SDS-eluted pulldowns (50% of the sample) was loaded for each GFP-Trap sample. (B) The 2D CN-SDS/PAGE analysis of the GFP-Trap pulldowns from the PSI-YFP and WT strains eluted by low pH (glycine buffer pH = 2.5) and immediately neutralized by Tris buffer. A total of 15 μL of low pH-eluted pulldowns (50% of the sample) was analyzed by CN-PAGE and lanes were cut and reelectrophoresed in the second dimension. The 2D gels were stained by SYPRO Orange, blotted to a PVDF membrane, and Ycf48 and D1 were consecutively detected using specific antibodies (segment of the blot containing individual antibody signals is shown).