Western Blot: stripping of the membrane
| Materials | |
|---|---|
| Mild Stripping Buffer | 15 g Glycin, 1 g SDS, 10 ml Tween20 in 1L
ddH2O pH2.2 |
| Harsh Stripping Buffer | 40 ml Stacking gel buffer (0,5M Tirs/HCl, pH6,8)
10 ml SDS 20%, 200 ml ddH2O |
| 1X PBS | |
| TBST | |
| ⚠️ for stripping use the old, already blue stained boxes ⚠️ | |
Procedure mild stripping
- Remove ECL from membrane by washing membrane briefly with ddH2O
- Wash membrane 2x in Mild stripping buffer for 10 min
- Wash membrane 2x in PBS for 10 min
- Wash membrane 2x for 5 min in TBS-T
- Block in Blotto for 1h at RT
- Apply 1st AB overnight, at 4°C
Procedure harsh stripping
- Prewarm water bath to 50°C
- Remove ECL from membrane by washing membrane briefly with ddH2O
- Add 10 ml of Harsh stripping buffer and supplement with 70 µl β- Mercaptoethanol
- Incubate the membrane for 20 min at 50°C, while shaking
- Wash the membrane under running tap water
- Wash for 1h with ddH2O, change ddH2O every 10 min
- Wash for 1h with TBS-T, change TBS-T every 10 min
- Block in Blocking buffer (Blotto) at least for 1h at RT
- now the membrane is ready for another round of probing