9). 100 mg of the isolated cell
wall material was then added to this solution and incubated over night at 28°C. The sample was then centrifuged and the pellet discarded. After heating (5 min; 100°C), centrifugation (10 min 10,000×g) and dialysis (molecular weight cut off 1000), the sample was freeze-dried. Resuspended lyophilized material was then used for further experiments. Removing LPS from the samples via polymyxin B agarose X. campestris pv. campestris lipopolysaccharides (LPSs) were removed from the elicitor preparation using a batch technique by adding an excess amount of polymyxin B agarose [102] as described in [103]. Upon addition of polymyxin B agarose (Sigma-Aldrich), the samples were shaken and centrifuged. While the pellet
probably containing LPS bound to polymyxin B agarose was discarded, the GW786034 cost supernatant was used for further analyses. Identification, isolation and characterization of oligosaccharides The SHP099 supplier analyses of oligosaccharides was performed by HPAEC using a DIONEX GP-40 gradient pump; a Merck-Hitachi D-2000 Chromato Integrator; a DIONEX pulsed amperometric detector and a DIONEX UV detector. Monosaccharide composition of isolated oligosaccharides was analyzed upon acid hydrolysis in trifluoroacetic acid (2 M; 120°C for 2 h). Neutral sugars were separated and identified using an isocratic elution (10 mM sodium hydroxide; flow 1 ml/min) with amperometric detection on a CarboPac® PA-100 column. For charged sugars a linear sodium acetate gradient ranging from 0.02 M to 0.5 M under alkaline conditions (0.1 M NaOH) with a flow rate of 1 ml/min was used [75]. Pectate fragments were separated using a sodium acetate gradient (ranging from 0.01 M to 1.0 M with a plateau of 10 min. at a concentration 0.7 M sodium acetate; 0.1
M NaOH; CarboPac® PA-100 column; flow 1 ml/min). For the identification of pectate fragments Plasmin a pectate standard was generated by digestion of pectin (Pectin esterified, Sigma P-9561) by pectate lyase (Sigma P-7052). The isolation of pectate fragments was carried out under the conditions described above, but a semi-preparative column (CarboPac® PA-1; flow 2.5 ml/min) was used. MALDI-TOF MS of isolated oligosaccharides Crude extracts were analyzed on a Bruker ultraflex I MALDI-TOF mass spectrometer (Bruker-Daltonics, Bremen, Germany) in the negative–ion mode. Samples were analyzed in the linear and in the reflector TOF. Gentisic acid was used as matrix. For sample preparation, 1 μl saturated gentisic acid solution was mixed with 1 μl of 50 mg ml–1 crude selleck chemicals extract lyophilisate dissolved in demineralized water. One microliter of this mixture was dropped onto the MALDI target. Determination of the oxidative burst reaction in plant cell suspension cultures The detection of the oxidative burst was performed using the H2O2-dependent chemiluminescence reaction described by Warm [104].