bl21  (New England Biolabs)

Journal: Plant and Cell Physiology

Article Title: A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha

doi: 10.1093/pcp/pcv160

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

Journal: bioRxiv

Article Title: Improve Protein Solubility and Activity based on Machine Learning Models

doi: 10.1101/817890

Figure Lengend Snippet: ( a) The SDS-PAGE analysis of protein tal and dxs expressed in E. coli with and without tags designed by our optimization algorithm. “+” and “-” represented expressed proteins with and without peptide tags respectively. “P” and “S” represented the pellet fraction (insoluble) and supernatant fraction (soluble), respectively. The oval shapes highlight the bands of dxs and tal proteins. Protein tal and dxs were expressed in K3 medium with 20 g/L glucose at 30 °C. ( b) Quantitative presentation of the SDS-PAGE images in a . The protein solubility was the ratio of soluble protein amount to the total protein amount. The protein amount was estimated by using band intensity on SDS-PAGE images. The sequences of the designed tags for N-terminal and C-terminal were shown. The amino acid S and G on the two ends of the tags were the linkers for GT DNA assembly standard, which was used to construct the plasmids in this study 40 .

Article Snippet: Cell culture and SDS-PAGE analysis of protein solubility Each of constructed plasmid was introduced into E. coli BL21 (DE3) (C2530H, New England Biolabs) for SDS-PAGE analysis by using standard heat shock protocol.

Techniques: SDS Page, Solubility, Construct

Journal: Journal of Clinical Microbiology

Article Title: Pan-Serotype Diagnostic for Foot-and-Mouth Disease Using the Consensus Antigen of Nonstructural Protein 3B

doi: 10.1128/JCM.03491-14

Figure Lengend Snippet: Recombinant c3B protein expression, purification, and detection. (A) Coomassie blue-stained polyacrylamide gel of recombinant MBP-c3B protein expressed in BL21 E. coli and affinity purified using amylose resin. Lane M, molecular mass markers (Mark 12;

Article Snippet: Chemically competent BL21 E. coli (NEB) was transformed with 10 ng pMAL-c5e or pMAL-c3B for overexpression of recombinant MBP or MBP-c3B fusion protein, according to the manufacturer's instructions.

Techniques: Recombinant, Expressing, Purification, Staining, Affinity Purification