Dot Blot
| Materials | |
|---|---|
| Nitrocellulose membrane / Whatman paper | |
| TBS-T | |
| Ponceau S | |
| Blocking Solution | |
| First and Second Antibody | |
| ECL solution and H2O2 |
Procedure
- Whatman paper and membrane have to be wet first whatman on dot blot, followed by membrane. Top of dot blot holder has to be firmly secured with screws on the lower part!
- Apply 50 µl of probe with equivalent amount of protein in dots
- Start vacuum till solution is through membrane
- Dry membrane for 10 min
- Stain membrane with Ponceau S for 1 min, short destain with TBS-T to check, if same amount of protein is on the blot, make a copy or scan blot
- Complete destaining of membrane with TBS-T
- Block the membrane for 2h with blotto at room temperature or over night at 4°C
- 1. antibody, over night at 4°C or 2h at room temperature
- Wash 3x for 10 min at room temperature with TBS-T
- 2. antibody, 1h at room temperature
- Wash 3x for 10 min at room temperature with TBS-T
- 10 ml ECL solution + 3 µl H2O2 for 2min at room temperature
- Develop membrane