The extracted lipids (30 µL) were spotted onto TLC plates (Yantai Jiangyou silicone company) and developed with neutral lipids developing buffer (n-hexane:ether:acetic acid = 80 20 1, v/v/v).
for an hour and then incubated with developing buffer (Invitrogen Corp).
The gel was then further incubated overnight (16 20 hr) at 37°C in the same developing buffer.
After washing three times 10 minutes with TBS, the membranes were equilibrated to developing buffer (100 mM NaCl, 100 mM Tris-HCl, pH 9.5) and developed in the dark with 100 µl BCIP and 100 µl NBT in 100 ml developing buffer until antigen spots became visible.
After washing three times for 10 minutes with TBS, the membranes were equilibrated to developing buffer (100 mM NaCl, 100 mM Tris-HCl, pH 9.5) and developed in the dark with 100 µl BCIP and 100 µl NBT in 100 ml developing buffer until antigen spots became visible.
After separation, the gel was re-naturated in 2.5% Triton X-100 solution at room temperature for 60 min with gentle agitation, equilibrated in developing buffer (50 mM Tris-HCl, pH 7.4; 200 mM NaCl, 5 mM CaCl2, 0.02% Brij35) at room temperature for 30 min with gentle agitation, and incubated overnight at 37°C in fresh developing buffer.
After washing three times for 10 minutes with TBS, the membranes were equilibrated to developing buffer (100 mM NaCl, 100 mM Tris-HCl, pH 9.5) and developed in the dark with 100 µl BCIP and 100 µl NBT in 100 ml developing buffer until antigen spots are visible.
After electrophoresis gels were washed in renaturing buffer (Novex) containing Triton X-100 to remove any SDS and incubated in developing buffer (Novex) for 18 hours at 37°C.
Gels were switched to developing buffer, pH 7.5 (50 mM Tris, 0.2 M NaCl, 5 mM CaCl2, 0.02% Brij-35) for two, 30-minute washes at room temperature and 24 hours at 37°C.
Plates were washed 4 times and antibody reactivities detected with 100 µl of substrate (0.4mgml-1 o-phenylenediamineine; 0.08% H2O2) in developing buffer (24.5 mM citric acid monohydrate; 52 mM Na2HPO4, pH 5.0).
Gels were renatured by washing for 2×30 min at 22°C in zymogram renaturing buffer followed by washing for 2×20 min in zymogram developing buffer (both obtained from InVitrogen) and incubation in the same buffer for 48 h.
