FIG. 1.

Strategy to isolate PAP I-GFP complexes. An E. coli strain that expressed a GFP-tagged PAP I was constructed as described in Materials and Methods. PAP I-GFP was visualized by fluorescence microscopy to determine intracellular localization. Cells were frozen and subjected to cryogenic cell lysis, which allowed for efficient lysis of >95% of cells, as determined by light microscopy. Proteins were isolated on magnetic beads coated with high-purity anti-GFP antibodies, resolved by SDS-PAGE, and identified by MS and MS/MS analyses using a MALDI LTQ Orbitrap mass spectrometer.