- Proteins and Peptides
- Lysates and Cell Lines
Positive Control Samples:
(1) HeLa cells were treated with 100M CdCl2 for 2 hours at 37C. The media was changed (i.e. growth
media without CdCl2) and the cells were allowed to recover for 5 hours at 37C. Cells were washed with
PBS and then harvested. Cells were collected from media by centrifuging at 1200 rpm at 4C in a
Beckman GS-6R centrifuge for 7 minutes. Cell pellets were resuspended with PBS+0.05% Triton-
X100+protease inhibitors (aprotinin, leupeptin, pepstatin) and then sonicated. The protein
concentration was determined and the sample was then diluted in SDS-PAGE buffer (40 mM Tris-HCl pH
6.8, 1% SDS, 50 mM DTT, 7.5% glycerol, 0.003% bromophenol blue) and heated at 70C for 5 minutes.
(2) Rehydrate rabbit liver MTI/MTII (Sigma cat# M7641) with 50mM Tris pH 5.5/50mM betamercaptoethanol.
Rehydrate the other MT proteins from Sigma with 10mM Tris pH 5.5/50mM betamercaptoethanol.
Proteins were prepared for gel loading by diluting in 5X sample buffer and boiling for
Western blotting protocol:
SDS-PAGE: 4-15% gradient gel (Biorad)
Transfer to PVDF membrane
Block membrane with 5% skim milk/0.05%T20/0.02% Thimerosal/PBS overnight at room temperature.
Incubate with #56262 at 1 ug/ml in 5 mls of Pierce's Superblock for 1 hr at room temp.
Wash 2 X with PBS/0.05% T20 for 5 min.
Incubate with secondary antibody in Pierce Superblock (Pierce cat# 37515) for 1 hr at room temp.
Wash 3 X PBS/0.05% T20 for 5 minutes
Detection with Amersham's ECL kit - exposure 2 min, Developer 1 min.