2D membrane and 2D Western blot:
Mouse liver samples were prepared using 2D lysis buffer. 100
ug of proteins was labeled and used for 2D electrophoresis or
2D DIGE using pH 4-7 linear IPG strip according to Applied Biomics
protocol. Left: standard 2D gel; Right: 2D DIGE gel. Following
2D electrophoresis, the images were scanned using Typhoon 9400;
then the 2D gel was subject to transfer to membrane; after protein
transfer, the 2D membrane images were scanned again; and followed
by double-color Western blot (left) to reveal keratin 8 (red)
and keratin 18 (green) including their modified isoforms by
CF555-labeled goat anti-rabbit Ig and CF647-labeled goat anti-mouse
Ig (Biotium, Hayward, California); after the scanning, the 2D
membrane was stripped and re-blocked followed by Western blot
to reveal beta-actin. For 2D DIGE gel (right panels) membrane,
using mouse anti-human keratin 8 to reveal all the K8 and its
posttranslational modified forms (under the disease condition);
secondary antibody is CF488-labeled goat anti-mouse IgG from
Biotium.
......
