The second part of the protein pellet (1 mg RJ/100 μl buffer) was suspended in a coupling buffer [100 mM sodium acetate (Sigma), 150 mM NaCl (Sigma), pH 5.5] and then prepared by enriching the N-linked glycopeptides with hydrazide resin according to the method of Zhang et al. [ 54].
The remaining third of the protein pellets (1 mg RJ/100 μl buffer) was suspended in 8 M of urea in 100 mM of Tris-HCl (pH 8.5) and the mixture was transferred into an Ultracel YM-10 10,000 MWCO centrifugal filter unit (Millipore) and digested by trypsin overnight.
After washing four times with RIPA buffer, beads were suspended in 20 μl 2 × SDS loading buffer, boiled at 100 °C for 10 min, and analyzed by western blot.
After an additional wash in ELB buffer, the pellets were suspended in ELB buffer and sonicated.
After washing thrice with Apoptosis Wash Buffer, the cells were suspended in 300 μl buffers and analyzed with a fluorescence microscope in three independent experiments.
After centrifuging and washing with buffer A, cells were suspended in 500 μL of buffer A and incubated in the dark for 15 min with 0.05 mM dihydroethidine (DHE).
After a final wash in low salt (150 mM) buffer, the beads were suspended in 20 μl of low salt wash buffer and SDS loading dye.
After 5 washings with 150 µl of the binding buffer, the spores were suspended in 300 µl of 50 mM Tris-HCl, pH 7.5, and incubated with 20 units of uPA and substrate.
After centrifugation and washing three times with blocking buffer, the cells were suspended in UV-irradiated PBS and used for epifluorescence microscopy or flow cytometry.
Then, the samples were washed with 2 mL permeabilization buffer, and the cells were suspended in Macs buffer for flow cytometric analysis [15].
