primary antibodies against e coli  (Bio-Rad)

Journal: iScience

Article Title: Metabolic silencing induced by the small bacterial membrane protein YohP

doi: 10.1016/j.isci.2025.114123

Figure Lengend Snippet: Native YohP levels increase when cells enter stationary phase or grow on minimal media (A) E. coli GSO33, an MG1655 variant containing a chromosomally SPA-tagged YohP under its native promoter (YohP-SPA), was grown on LB medium up to different ODs, and 2 × 10 8 cells were precipitated with 5% trichloroacetic acid (TCA). After centrifugation, samples were denatured and separated on 16.5% Tris-Tricine SDS-PAGE followed by western transfer and immune detection with α-FLAG antibodies. The inner membrane protein YidC served as a loading control and was detected by polyclonal α-YidC antibodies. (B) MG1655 (WT) and its variant GSO33 were grown on LB or M63 minimal medium up to OD 600 = 1.5, and cells were processed as above. Before blocking, the membrane was stained with Ponceau Red to control the amount of loaded protein. (C) MG1655 and GSO33 cells were grown on LB medium +0.2% glucose in either completely filled 2 mL Eppendorf tubes without shaking (oxygen-limited) or in 50 mL Falcon tubes with 5 mL LB + 0.2% glucose medium with shaking (aerobic). Cells were harvested at OD 600 = 1.5 and processed as described above. (D) GSO033 cells were grown on LB medium, and at OD 600 = 0.2 SDS (0.025%) and EDTA (1 mM) were added when indicated. After growth to OD 600 = 1.5, cells were processed as above. (E) GSO33 cells were grown on LB medium and at OD 600 = 0.2, 1 mM H 2 O 2 was added. After growth to OD 600 = 1.5 cells were processed as described above. Shown are representative blots of at least 3 independent biological replicates. See also .

Article Snippet: rabbit α-TnaA (polyclonal) , AssayPro, St.Charles, USA , Cat#33517-05111.

Techniques: Variant Assay, Centrifugation, SDS Page, Western Blot, Membrane, Control, Blocking Assay, Staining

Journal: iScience

Article Title: Metabolic silencing induced by the small bacterial membrane protein YohP

doi: 10.1016/j.isci.2025.114123

Figure Lengend Snippet: Premature production of YohP in exponential phase inhibits E. coli cell growth (A) E. coli MG1655 carrying the chromosomally tagged yohP copy (GSO33) was grown on LB medium up to OD 600 = 2.5 (stationary phase, stat.). E. coli MG1655 cells containing the plasmid-encoded yohP variants in either vectors pRS1 or pBad24 were induced at OD 0.4 with either 1 mM IPTG or 0.2% arabinose for approximately 1–2 h (OD 600 ∼1.0, exponential phase, exp.). The Δ yohP strain served as a control. 2 × 10 8 cells were precipitated with 5% trichloroacetic acid (TCA). After centrifugation, samples were denatured and separated on 16.5% Tris-Tricine SDS-PAGE, followed by western transfer and immune detection with α-FLAG antibodies. The inner membrane protein YidC served as a loading control and was detected by polyclonal α-YidC antibodies. (B) MG1665 carrying pRS1-YohP(FLAG) 3 was grown on LB medium up to the indicated OD 600 ; YohP production was induced at OD 600 = 0.2 with 1 mM IPTG. 2 × 10 8 cells were precipitated with 5% trichloroacetic acid (TCA) and further processed as above. Representative blots of three independent experiments are shown. (C) The indicated strains were grown on LB medium, and YohP production from the plasmid was induced at OD 600 0.2 with either 1 mM IPTG or 0.002% arabinose. Cell growth was then monitored over time. Shown are the mean optical density readings of three independent biological experiments, and the error bars reflect the SD. See also .

Article Snippet: rabbit α-TnaA (polyclonal) , AssayPro, St.Charles, USA , Cat#33517-05111.

Techniques: Plasmid Preparation, Control, Centrifugation, SDS Page, Western Blot, Membrane

Journal: iScience

Article Title: Metabolic silencing induced by the small bacterial membrane protein YohP

doi: 10.1016/j.isci.2025.114123

Figure Lengend Snippet: YohP causes membrane damage (A) E. coli cells were grown on LB medium up to an OD of 1.3, and 2 × 10 8 cells were processed as in and the membrane was decorated with the indicated polyclonal antibodies, or, in the case of YohP, with monoclonal α-His antibodies. YohP production was induced with 1 mM IPTG at OD 600 = 0.4. Representative blots of three independent experiments are shown. (B) Lipidomic analyses of isolated inner membrane vesicles (INVs) derived from the indicated strains. The extraction, analysis, and quantification were performed by Lipotype GmbH, Dresden, Germany, as described in STAR Methods. Shown are the percentage of the main E. coli lipids (PE, phosphatidylethanolamine; PG, phosphatidylglycerol; and CL, cardiolipin). The individual values are indicated by dots, the mean values by the columns, and the SD by the error bars ( n = 3). (C) Membrane order in INVs of the indicated strains (1 mg protein/ml) was monitored in terms of the limiting fluorescence anisotropy of the probe 1.6-Diphenyl-1,3,5-hexatriene (DPH), i.e., the value approached by the anisotropy decay for infinite time after excitation. Shown are scatterplots of four independent experiments, with the mean value indicated by the column and the SD by the error bar. To determine the significance of the results, the P-value was calculated using the “unpaired, two-tailed t test” of the program Graph Pad PRISM 6 or the one-way ANOVA analyses and Turkey honest significance test using the values in the wild-type strain as reference. The P-values are depicted as asterisks (∗) above the graphs as follows: n.s. = p > 0.05; ∗ = p ≤ 0.05; ∗∗ = p ≤ 0.01. See also .

Article Snippet: rabbit α-TnaA (polyclonal) , AssayPro, St.Charles, USA , Cat#33517-05111.

Techniques: Membrane, Isolation, Derivative Assay, Extraction, Fluorescence, Two Tailed Test