Hptlc silica gel 60 plate

The enzyme assay was performed using 20 ml E. coli cultures expressing the respective constructs right after induction with L-arabinose. 100 μl substrate [10 mg/ml Naringenin, eriodictyol or dihydroquercetin (DHQ)], 50 μl 2-oxoglutaric acid, 50 μl FeSo₄ and 50 μl 1 M sodium ascorbate were added. The cultures were incubated at 28°C overnight. To extract flavonoids, 1 ml was removed from each culture and mixed with 200 μl ethyl acetate by vortexing for 30 s. Samples were taken after 0 h, 1 h, 2 h, 3 h, 4 h and 24 h. After centrifugation for 2 min at 14,000 g, the organic phase was transferred into a fresh reaction tube. Flavonoid content was analysed by high-performance thin-layer chromatography (HPTLC). Naringenin (Sigma), dihydrokaempferol (DHK; Sigma), kaempferol (Roth), eriodictyol (TransMIT PlantMetaChem), apigenin (TransMIT PlantMetaChem), DHQ (Roth) and quercetin (Sigma) were dissolved in methanol and used as standards. 3 μl of each methanolic extract was spotted on a HPTLC Silica Gel 60 plate (Merck). The mobile phase was composed of chloroform, acetic acid and water mixed in the ratio (50:45:5). Flavonoid compounds were detected as described previously (Stracke et al., 2007 (link)), using diphenylboric acid 2-aminoethyl ester (DPBA) and UV light (Sheahan and Rechnitz, 1992 (link)).

Radiolabeled LLSs of induced E. coli CS2775 cells bearing the selected plasmids were extracted using CHCl₃/CH₃OH and water-saturated butan-1-ol (1:1 v/v) solution to determine the addition of sugar residues in vivo after glucose D[6s³H] (N) (PerkinElmer Life Sciences) supplementation (1 mCi/ml). The incorporated radioactivity was measured in a Beckman LS6000SE scintillation counter. The organic phase containing the LLSs was normalized to 0.05 μCi/μl. The samples were separated via TLC on an HPTLC Silica Gel 60 plate (Merck) using a C:M:AC:A:W mobile phase (180 ml of chloroform + 140 ml of methanol + 9 ml of 1 m ammonium acetate + 9 ml of 13 m ammonia solution, 23 ml of distilled water), the plates were dried and sprayed with En3Hance liquid (PerkinElmer Life Sciences). Radioautography images were obtained using Carestream® Kodak® BioMax® XAR Film and MS intensifying screens after 5–10 days.

Phospholipids of A. tumefaciens or E. coli strains were isolated according to Bligh and Dyer [23 (link)]. Briefly, 1 to 2 ml of culture were harvested by centrifugation, washed with 500 μl of water to remove residues of medium and resuspended in 100 μl of water. 375 μl of a mixture of methanol:chloroform (2:1) was added and samples were homogenized before 100 μl of water and 100 μl of chloroform were added. Samples were briefly vortexed and centrifuged for 5 minutes at 13.000 rpm. The lower organic phase was collected and dried under vacuum. The lipid pellet was resuspended in 15 μl of methanol:chloroform (1:1) and spotted onto a HPTLC silica gel 60 plate (Merck, Darmstadt, Germany). For 1D-TLC a mixture of n-propanol:propionate:chloroform:water (3:2:2:1) was used as a running solvent. For two-dimensional TLC (2D-TLC), mixtures of chloroform:methanol:water (65:25:4) and chloroform:methanol:acetic acid:water (90:15:10:3,5) were used as running solvents for first and second dimension, respectively. For the visualization of the lipids, plates were sprayed with molybdenum blue reagent (Sigma-Aldrich, St. Louis, MO, USA) or charred after CuSO₄-treatment at 180°C. Purified phospholipid standards (bovine heart cardiolipin, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol), L-α-phosphatidylethanolamine) were purchased from Merck (Darmstadt, Germany).

To extract lipids, 0.5 ml of protein solution or buffer only was mixed with 1.9 ml of chloroform:methanol = 1:2 (v/v) mixture in a glass tube (chloroform:methanol:water = 1:2:0.8 (v/v/v))^{62 (link)}. The mixture was vortexed and incubated at room temperature for 10 min. After incubation, we added 0.6 ml of chloroform and 0.6 ml of water and mixed before centrifugation. We collected the bottom organic phase in another fresh glass tube and added 0.9 ml chloroform to the remaining aqueous solvent, followed by mixing and centrifugation. The bottom phase was collected and mixed with the first organic phase. After evaporation, the extracted lipids were dissolved in a small volume of chloroform and spotted 1 cm from the lower edge of an HPTLC Silica gel 60 plate (Millipore). The plate was placed in a chamber containing a developing solvent composed of chloroform:methanol:water = 65:25:4 (v/v/v) and incubated until the running front of the solvent was close to the upper edge. After incubation and air-drying, the plate was sprayed with H₂SO₄ and baked until the lipids became visible. As standards, l-α-phosphatidylethanolamine from egg yolk (Sigma-Aldrich) and l-α-phosphatidyl-dl-glycerol from egg yolk (Sigma-Aldrich) were developed in the same plate.

The digestion of Lys-type lipid II (gift from Eefjan Breukink, University of Utrecht) was assessed by thin-layer chromatography as previously described [46] . Briefly, TelC_tox alone or TelC_tox with 1.2 molar equivalents of TipC1_ΔTMD, TipC1_ΔTMD^F71Q, TipC1_ΔTMD^K93E or TipC1_ΔTMD^{F71Q, K93E} was incubated in a total volume of 50 μl with 2 nmol lipid II in 150 mM KCl, 0.1% Triton X-100 and 2 mM CaCl₂ for 90 min at 37 °C. Lipids were extracted with n-butanol/pyridine acetate (2:1) at pH 4.2 and resolved on an HPTLC silica gel 60 plate (Millipore) developed with chloroform/methanol/ammonia/water (88:48:1:10). Compounds were stained with iodine, and bands were quantified by the ImageJ software.