Western blot for detecting decorin-binding capability of dbpA mutants
(created by Qilong Xu on 2008-07-22 (updated at 2008-07-27 20:23:24)
Reagents
Biotin labeled decorin, 10% Blocker BSA, ImmunoPure Streptavidin Horserodish Peroxidase Conjugate, Towbin transfer buffer/ methanol, W.B. reagents.
1x PBST: 1x PBS with 0.05% Tween20
Procedures
1. Harvest 1ml cells (ΔdbpAB-fA, ΔdbpAB-fAm1, ΔdbpAB-fAm3) cultured to stationary phase, wash twice with 1x PBS buffer. Resuspend with 20 µl 1X PBS and 10µl of loading dye. Total volume is 30 µl, as loading mix.
2. Load 15 µl - 20 µl of the loading mix to 12% SDS-PAGE gel. Run 30 min at 80 Voltage, then about 1 hour at 100 Volt.
3. Membrane transfer through 1x Towbin buffer for 1 hour at 300 mA.
4. Blocking membrane in 7 ml of 1% BSA in PBST buffer for 1 hour.
5. During the course of membrane blocking, add 8 µl of biotin-decorin to 0.7 ml of 10% BSA. Incubate at room temperature for 1 hour. (Note: spin down biotin-decorin and use supernatant if any precipitation)
6. Transfer the membrane to 6.3 ml of 1x PBST buffer with the mixture (~0.7ml) from step 5. Incubate at room temperature for 1~2 hours or 4ºC overnight.
7. Wash membrane with 1x PBST buffer for 3 times x 3 min.
8. Incubated membrane in 7 ml of 1x PBST buffer with 5 µl of strepavidin-conjugated secondary antibody for 1 hour.
9. Wash membrane as step 7.
10. Development membrane with W.B. reagents.