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. 2019 Apr 5;8:e42475. doi: 10.7554/eLife.42475

Figure 2. Production of PhyB tetramers.

Figure 2.

(A) Schematics of the PhyB1-651 construct and the PhyB tetramers. PCB = phycocyanobilin. (B) Purified PhyB was illuminated with 660 nm light [PhyB(660)] and added in a 1:2 molar ratio to MBP-PIF(wt). The proteins were separated by gel filtration and PhyB was detected by its absorbance at the isosbestic point of 671 nm. PhyB molecules around 14.5 ml elution volume are the free PhyB molecules and the ones around 12.5 ml are the PhyB-MBP-PIF complexes. As controls, PhyB illuminated with 740 nm light [PhyB(740)] plus MBP-PIF(wt) and PhyB alone was only detected at 14.5 ml elution volume. Results show one experiment of n > 3. (C) Affinity chromatography-purified PhyB was mixed in a 10:1 molar ratio with streptavidin-DyLight650, incubated for 2 hr at room temperature and the formed PhyB tetramers (PhyBt) were isolated from monomers using size-exclusion chromatography. The elution of PhyB was monitored via its absorbance at 365 nm. Results show one experiment of n > 3.