storm phosphorimager  (Molecular Dynamics Inc)

Journal: ACS Omega

Article Title: Fluorous-Directed Clamping Stabilizes Triple-Helical DNA

doi: 10.1021/acsomega.6c00803

Figure Lengend Snippet: Triplex formation by a fluorous-modified TFO. DNase I cleavage patterns of a 73-mer duplex containing the TFO target sequence in the absence and presence of ODN1 and ODN2 ( Figure S1B ). Assays were undertaken at pH 5.0 in sodium acetate buffer containing 10 mM MgCl 2 and the complexes incubated overnight at 4 °C before digestion by the enzyme. Final TFO concentrations were 3, 1, and 0.3 μM in lanes 2, 3, and 4, respectively. The products of the reaction were separated on a 12% denaturing polyacrylamide gel and visualized via phosphorimaging. The TFO target sequence is indicated by the black boxes and was determined by reference to bands in the Maxim–Gilbert “marker” lane (lane 0). The full nucleotide sequence is shown on the left of the gel, and the observed purines are shown in bold.

Article Snippet: Polyacrylamide gels were run at 1500 V for ∼2 h, fixed in 10% (v/v) acetic acid, transferred to Whatman 3MM paper, and dried under vacuum at 86 °C for 1 h. The dried gels were subjected to phosphorimaging using a Molecular Dynamics Typhoon PhosphorImager.

Techniques: Modification, Sequencing, Incubation, Marker

Journal: mBio

Article Title: Lysophospholipid signaling coordinates outer membrane homeostasis in Escherichia coli

doi: 10.1128/mbio.00567-26

Figure Lengend Snippet: PF resistance and reduced envelope stress in LPL-recycling mutants are PldA-dependent. ( A ) Efficiency of plating assays comparing WT and LPL-recycling mutants lacking PldA in the absence or presence of 25 ng/mL PF. ( B ) LPS profiles of WT and ∆ pldA strains grown with PF. Cultures were grown to an OD 600 of 0.4 and exposed to 40 ng/mL PF for 4 h, and changes in LPS were assessed by SDS-PAGE. Capsule addition is indicated by a bracket. ( C ) TLC of extracted GPL species from WT or ∆ pldA backgrounds with (red) and without (black) PF treatment. Following radiolabeling, GPLs were isolated and separated in the solvent chloroform:methanol:acetic acid (65:25:10, vol/vol/vol). Lipids (20,000 cpm per lane) were visualized by phosphorimaging, and densitometry within each lane was used to determine the percentage of lyso-PE present. Changes in lipid composition were similar for other biological replicates. Data are representative of a minimum of three biological replicates.

Article Snippet: Plates were exposed to a phosphorimaging screen overnight and imaged using the Amersham Typhoon Biomolecular Imager (Cytiva).

Techniques: SDS Page, Radioactivity, Isolation, Solvent

Journal: mBio

Article Title: Lysophospholipid signaling coordinates outer membrane homeostasis in Escherichia coli

doi: 10.1128/mbio.00567-26

Figure Lengend Snippet: Loss of LPL recycling improves OM asymmetry during LpxC inhibition. ( A ) TLC of lipid A species from WT and ∆ pldA backgrounds grown with (red) and without (black) PF. Each sample (5,000 cpms) was separated in the solvent chloroform:pyridine:88% formic acid:water (50:50:16:5, vol/vol/vol/vol). Lipids were visualized by phosphorimaging, and densitometry was used to calculate the percentage of hepta-acylated lipid A (i.e., PagP activity). Changes in lipid composition were similar for other biological replicates. ( B ) GPL-to-LPS ratios in the absence and presence of PF. Cultures were grown to an OD 600 of 0.4 and exposed to 40 ng/mL PF for 2 h. GPLs and lipid A were extracted and quantified by scintillation counting. ( C ) Quantification of GPL and lipid A production following PF treatment. The cpms for each lipid sample were normalized to the total amount of 32 P incorporation into the cell. GPL and lipid A levels relative to replicate 1 of WT are shown. Data for GPL-to-LPS ratios is from five biological replicates. ( D ) GPL synthesis assay during LpxC inhibition. Cells were grown with 32 P i to an OD 600 of 0.4, when 40 ng/mL PF was spiked into cultures. At 30-min intervals over a 2 h period, aliquots were taken for GPL extraction and for the determination of total cellular 32 P incorporation. Counts from extracted GPLs were first normalized to total radiolabel incorporation and then compared to WT GPL levels at 0 min and plotted over time. Data are representative of three biological replicates. ( E ) Efficiency of plating assays assessing changes in OM permeability. Cultures were grown to an OD 600 of 0.4, treated with 30 ng/mL PF for 2 h, washed, and normalized by OD 600 . Serial dilutions were spotted on the indicated LB agar plates, and growth was evaluated. Error bars represent SD for bar graphs (** P -value ≤ 0.01; **** P -value < 0.0001). Data are representative of a minimum of three biological replicates.

Article Snippet: Plates were exposed to a phosphorimaging screen overnight and imaged using the Amersham Typhoon Biomolecular Imager (Cytiva).

Techniques: Inhibition, Solvent, Activity Assay, Extraction, Permeability