Journal: Plant and Cell Physiology
Article Title: A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha
Figure Lengend Snippet: Detection of chloroplast-expressed mTurquoise2 in transgenic lines of Marchantia polymorpha. (A) M. polymorpha protein extracts separated by SDS–PAGE. Protein extracts from wild type (WT) and transplastomic (CL0*b) lines of M. polymorpha were separated by 4–12% SDS–PAGE, and visualized by in-gel fluorescence (left) and subsequent Coomassie stain (right). The fluorescence image was generated using a custom imaging device described in the Materials and Methods for visualization of CFP bands (emission 486/10 nm, green) and marker (emission 540/10 nm, red). (B) Untagged and His 6 -tagged mTurquoise2 separated by SDS–PAGE. Untagged mTurquoise2 was expressed from plasmids pCS CL0*b ( psbA promoter) in BL21 E. coli and pCRB SREI (T7 promoter) in T7 Express E. coli. His 6 -tagged mTurquoise2 was expressed from plasmid pCRB SREI 6his (T7 promoter) and purified by affinity chromatography. The rightmost lanes contain 50 ng of unboiled and boiled purified protein, respectively. Visualization by in-gel fluorescence and Coomassie stain was conducted as described above. (C) Standard curve for quantification of mTurquoise2 based on in-gel fluorescence. Serial dilutions of 1 µg purified of mTurquoise2 were separated by 4–12% SDS–PAGE and the band intensity quantified by in-gel fluorescence as described in the Materials and Methods. Levels of mTurquoise2 extracted from transplastomic (CL0*b) M. polymorpha (red cross) were estimated by linear regression analysis on densities of fluorescent target bands as shown in (A). Error bars represent the SD of average band density between three different experiments.
Article Snippet: Visualization of proteins by in-gel fluorescence and coomassie staining For total protein extraction from M. polymorpha tissues, 50 mg (fresh weight) of thallus were ground by liquid nitrogen and the resulting powder vortexed in 200 µl of 2 × Tris–glycine for 30 s. For total protein extraction from BL21 or T7 Express competent E. coli (New England Biolabs), an aliquot of overnight culture containing approximately 0.5–1 × 109 cells was pelleted by centrifugation at 14,000 r.p.m. and 4°C for 1 min, resuspended in 50 µl of 2 × Tris–glycine SDS sample buffer and vortexed for 30 s. In both cases, 2-fold dilutions of crude extracts were centrifuged at 14,000 r.p.m. and 4°C for 20 min to remove cell debris, and unboiled (unless indicated otherwise) supernatants were directly loaded onto a NuPAGE Novex 4–12% Bis–Tris protein minigel (Life Technologies).
Techniques: Transgenic Assay, SDS Page, Fluorescence, Staining, Generated, Imaging, Marker, Plasmid Preparation, Purification, Affinity Chromatography