3 µl of the resulting lysate was added to each well of a 96 well plate, reaction buffer (per well, containing 25 µl of 0.4 mg/ml DTNB (dissolved in 1 M Tris-HCl pH 8.1), 7.5 µl of 10 mg/ml acetyl CoA (dissolved in acidified water pH 5.0) and 165 µl water) was added to each well.
The cells were then lysed by 100 µl of passive lysis buffer per well on a shaker for 2 hours, and lysates were centrifuged for supernatant collection.
Membranes were then aspired onto a glass fiber filtermat (Printed Filtermat B, Wallach, Turku, Finland), which had been preconditioned with 0.33% polyethylenimine for 30 min and then washed five times with 300 μL (per well) of wash buffer (50 mM HEPES, 500 mM NaCl, 0.1% BSA, buffered to pH 7.4 using 1 N NaOH), using a Mach II M Harvester 96 (Tomtec CE, Etten-Leur, The Netherlands).
Membranes were washed ten times with 150 μL (per well) of wash buffer.
After saturation for at least 4 h at room temperature with EIA buffer, 50 µL per well of serial dilutions of individual serum (1/5 to 1/125, in EIA buffer) was dispensed.
50 µL of beads, resuspended to 0.025% solids in 1x hybridization buffer, were added per well of a bead source plate.
After 4 washes with wash buffer, 200 μL per well of o-phenylenediamine hydrochloride solution (1 mg mL−1 in hydrogen peroxide substrate) was incubated for 10 20 min. The reaction was terminated by the addition of 50 μL per well 2 N H2SO4, and absorbance was measured on a plate spectrophotometer (Molecular Designs) at 490 nm.
After 24 hr the cells were washed in phosphate-buffered saline, lysed, and harvested by using 500 µl of Glo lysis buffer (Promega) per well.
Cells were washed once with PBS and lysed with 50 µl of cell lysis buffer (Promega) per well.
