Journal: Nature Communications

Article Title: Investigation of artificial cells containing the Par system for bacterial plasmid segregation and inheritance mimicry

doi: 10.1038/s41467-024-49412-9

Figure Lengend Snippet: a Giant unilamellar vesicles (GUV) containing the ParMRC system and PURE system. b parC -eGFP DNA segregation by the polymerization of ParM triggered by adenosine triphosphate (ATP) influx upon laser irradiation. c A deformed GUV under hypertonic conditions. d Two daughter GUVs containing parC -eGFP DNA and the PURE system upon laser irradiation at the center region of the deformed GUV (purple area in Fig. 1c). e Enhanced green fluorescent protein (eGFP) was expressed inside two daughter GUVs through translating the eGFP gene using a PURE (protein synthesis using recombinant elements) system at 37 °C. The PURE system contains ribosomes, amino acids, nucleoside triphosphates (NTPs), transfer ribonucleic acid (tRNAs), enzyme substrates, RNA polymerase, translation factors, and other necessary components.

Article Snippet: A Bradford protein assay kit, SDS‒PAGE gel configuration kit, 4×SDS‒PAGE sample loading buffer, protease inhibitor cocktail for purification of His-tagged proteins, green fluorescent DNA marker dye, streptavidin magnetic beads, Tris-borate-EDTA buffer (TBE) and Tween-20 were purchased from Beyotime (China).

Techniques: Irradiation, Recombinant

Journal: Nature Communications

Article Title: Investigation of artificial cells containing the Par system for bacterial plasmid segregation and inheritance mimicry

doi: 10.1038/s41467-024-49412-9

Figure Lengend Snippet: a Schematic illustration of two parC -eGFP DNAs being pushed through ParM polymerization in a giant unilamellar vesicles (GUV) and subsequent GUV division under laser irradiation. b Confocal microscopy images of GUV division ( b1 – b3 ) and enhanced green fluorescent protein (eGFP) expression at 37 °C ( b4 – b7 ). b1 – b3 indicate GUV deformation, filament splitting, and division into two daughter cells, respectively. The white arrows in b1 and b2 indicate parC -eGFP DNA. The ParM filament was split by laser irradiation (561 nm, 0.7 mW, 5 s). The scale bars are 10 μm. n = 3 independent replicates. c Schematic illustration of eGFP expression in two daughter cells. The corresponding fluorescence intensity (FI) in daughter GUV 1 ( d ) and daughter GUV 2 ( e ) as a function of time. The normalized fluorescence intensity of eGFP was calculated from three independent samples. The data were presented as the mean values ± SDs; n = 3 independent replicates. Source data are provided as a Source Data file.

Article Snippet: A Bradford protein assay kit, SDS‒PAGE gel configuration kit, 4×SDS‒PAGE sample loading buffer, protease inhibitor cocktail for purification of His-tagged proteins, green fluorescent DNA marker dye, streptavidin magnetic beads, Tris-borate-EDTA buffer (TBE) and Tween-20 were purchased from Beyotime (China).

Techniques: Irradiation, Confocal Microscopy, Expressing, Fluorescence

Journal: Journal of Lipid Research

Article Title: Differential activity-based gel electrophoresis for comparative analysis of lipolytic and esterolytic activities

doi: 10.1194/jlr.M800566-JLR200

Figure Lengend Snippet: Optimization of time-dependent enzyme labeling by Cy-tagged ARPs. A–D: Homogenized brown adipose tissue (15 μg protein /lane) was spiked with CVL (150 ng/15 μg; highlighted by frames) as reference enzyme as described (see Experimental Procedures) followed by incubation with fluorescent ARP (A: 4a; B: 4b; C: 4c, final label concentration was 10 μM; D: whole protein stain using SYPRO Ruby). Incubation times were 5 min (1), 10 min (2), 30 min (3), 60 min (4), 90 min (5), 120 min (6), and 150 min (7). The reaction was stopped by precipitation of the protein with trichloroacetic acid followed by SDS gel electrophoresis. E–H: Effect of probe concentration on enzyme labeling by Cy-tagged ARPs. Mouse liver homogenate (15 μg/lane) was spiked with CVL as reference enzyme (150 ng/15 μg) followed by labeling with fluorescent ARP (E, 4a; F, 4b; G, 4c; H, SYPRO Ruby stain). Final probe concentrations were: 1, 0.5 μM; 2, 1.0 μM; 3, 2.5 μM; 4, 5.0 μM; 5, 7.5 μM; 6, 10.0 μM; 7, 15.0 μM; 8, 20.0 μM; 9, 25.0 μM. I–L: Effect of protein concentration on enzyme labeling by Cy-tagged ARPs. Mouse liver homogenate was spiked with CVL as reference enzyme (150 ng/15 μg) and labeled with fluorescent ARPs [I, 4a; J, 4b; K, 4c (10 μM final concentration); L, SYPRO Ruby stain]. The total protein contents of the samples were: 1, 0.5 μg; 2, 1.0 μg; 3, 2.0 μg; 4, 4.0 μg; 5, 6.0 μg; 6, 8.0 μg; 7, 10.0 μg; 8, 12.0 μg; 9, 15.0 μg.

Article Snippet: Fluorescent dye and inhibitor synthesis Organic solvents were obtained from Carl Roth (Karlsruhe, Germany).

Techniques: Labeling, Incubation, Concentration Assay, Staining, SDS-Gel, Electrophoresis, Protein Concentration

Journal: Journal of Lipid Research

Article Title: Differential activity-based gel electrophoresis for comparative analysis of lipolytic and esterolytic activities

doi: 10.1194/jlr.M800566-JLR200

Figure Lengend Snippet: DABGE of lipases in artificial proteomes. Three commercially available enzymes (CVL, GCL, and MME) were used to mimic a lipolytic proteome. Two separate enzyme mixtures were prepared differing in the CVL/GCL ratio. The amount of MME was the same in both samples. The ratios of lipases in sample A versus B were as follows: CVL 1:3, GCL 2:1, and MME 1:1. The individual samples A and B were labeled with the Cy3 and Cy5 probes, respectively. Independently, a mixture of samples A and B containing the same amount of total protein was labeled with Cy2b inhibitor. Finally, equal amounts of the Cy3-, Cy5-, and Cy2b-labeled aliquots were mixed and the resultant mixture was subjected to 2D-PAGE. In a dye-swap experiment, the same procedure was performed except that sample A and sample B were labeled with the Cy5- and the Cy3-ARP, respectively. A: Original experiment. B: Dye-swap. C: Quantitative analysis of the fluorescent intensities of the labeled lipases showed that the obtained enzyme ratios very closely reflected the amounts of the respective labeled enzymes.

Article Snippet: Fluorescent dye and inhibitor synthesis Organic solvents were obtained from Carl Roth (Karlsruhe, Germany).

Techniques: Labeling

Journal: Journal of Lipid Research

Article Title: Differential activity-based gel electrophoresis for comparative analysis of lipolytic and esterolytic activities

doi: 10.1194/jlr.M800566-JLR200

Figure Lengend Snippet: DABGE analysis of lipolytic enzymes in BAT and WAT. Fluorescence patterns of tagged proteins after 1D and 2D electrophoresis. A shows the results of the original experiment comparing BAT and WAT, whereas C shows the corresponding gel obtained after dye-swap. In a comparative experiment, proteins were separated by 2D gel electrophoresis leading to images B and D. The fluorescence images show that one fluorescent band of the 1D gel contains several different enzymes corresponding to several spots in the 2D image (e.g., lane n of the 1D PAGE contains the enzymes corresponding to the spots n1 and n2 in 2D PAGE). Higher amounts of active proteins in BAT as compared with WAT appear green in A and B (and red in C and D) and vice versa. Similar amounts in both tissues appear yellow. Relative enzyme ratios of BAT versus WAT and the protein identification are listed in Table1.

Article Snippet: Fluorescent dye and inhibitor synthesis Organic solvents were obtained from Carl Roth (Karlsruhe, Germany).

Techniques: Fluorescence, Two-Dimensional Gel Electrophoresis, Electrophoresis