Gel chemi doc

Recovered bacterial concentrations were estimated using the MPN Calculator, Build 23 (http://member.ync.net/mcuriale/mpn/index.html) based on 95% confidence intervals. The combined detection limits for the LC and HC assays used in Experiment A ranged from 1.13 MPN/head to 3.51 × 10⁷ MPN/head of E. coli Rif^r. The detection limits for Experiment B ranged from 341.6 MPN/head to 3.47 × 10¹² MPN/head of E. coli Rif^r. The raw MPN/mL or MPN/µL was extrapolated to a per head estimate by multiplying by the total washate volume, under the assumption that the washing techniques equally removed and homogenized the remaining E. coli Rif^r into the PBS-Rif washate.
Twenty percent of the presumptive positive colonies were randomly selected from positive ChromAgar ECC plates. Isolates were stored at −80 °C and characterized using pulse field gel electrophoresis (PFGE) to ensure that recovered bacteria were the inoculated strain and not rifampicin-resistant background E. coli in the lettuce field. Salmonella Braenderup strain H9812 was used as a control according to the CDC PulseNet standard laboratory procedure [35 (link)]. After completion of PFGE, the gel was stained with ethidium bromide, the image captured by Gel/Chemi Doc (Bio-Rad, Hercules, CA, USA), and the genetic similarity determined between different strains using GelCompar II software (Applied Maths, Austin, TX, USA).

Native-Polyacrilamide Gel Electrophoresis (Native-PAGE) was performed on PAGE (4.3% T; 7.3% C) with a running buffer composed of 4 mM Tris-HCl pH 8.3 and 38 mM glycine. In each lane of the gel, 200 μg of total proteins were loaded. After the electrophoretic run, the gels were washed with distilled water and incubated in specific buffers for the detection of APX and CAT, as described in Paciolla et al. [49 (link)]. For the SOD, the activity on the gel was visualized by incubating it in 0.053 Tris–HCl buffer pH 8.2 containing 0.21 mM riboflavin and 0.244 mM nitro-blue tetrazolium (NBT) in the dark; after 15 min, achromatic bands on a grey background appeared, a 50% glycerol solution was used to block the reaction.
For densitometric analysis of SOD activity, the gel was acquired utilizing the Gel/ChemiDoc and Quantity One software (BioRad Laboratories Inc., Milan, Italy) to obtain information on the changes in the activity of each band due to different treatments. A relative value of 100 was assigned to the intensity of the bands of Ctrl and I samples.