After washing and resuspending in media, the cells (usually 1×106 purified B cells per group) were incubated with various concentrations of the specified enterotoxin in media for 45 minutes at 37°C, washed in FACS buffer (PBS pH 7.4+1% FCS+0.005% NaN3), and then prepared for FRET or EM measurements, as detailed below.
Kidneys were surgically removed and then prepared.
And then prepare some more.
Phosphate buffer 1X, homogenization buffer and decontamination buffer (HDB) were then prepared from the kit for sample processing.
Briefly, LoVo cells were rinsed with ice-cold PBS, lysed using RIP lysis buffer, and then lysates were prepared.
To obtain total protein lysates, 80~90% of confluent cells was washed with chilled phosphate buffered saline (PBS), and cell lysates were then prepared on ice using cool lysis buffer (8 M urea, 4% CHAPS, 40 mM Tris, 1 mM EDTA, 1 mM EGTA, 60 mM DTT) containing a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany).
Additionally, one sample bearing only the reference standard was mixed with an equal volume of buffer A. These samples were then prepared for LC/MS via precipitation, reduction, alkylation, and tryptic digestion as described previously (Jomaa et al., 2014).
Human brain samples were prepared as 20% homogenates and equal protein concentrations then prepared for SDS PAGE by addition of SDS PAGE sample buffer and heating to 100°C.
The supernatant and pellet fractions were then prepared for analysis in lysis buffer as above prior to Western blotting.
A solution of 4 μg/mL methacryloxyethyl thiocarbamoyl rhodamine B was then prepared in carbonate buffer and contacted with the DTT functionalized gel for 30 min, followed by 30 min of washing in H2O.
