Table 3.
Western blotting parameters
| Antibody | Run time, hours/voltage | Transfer time hours | Blocking reagent/time, hours | Secondary antibody incubation, hours |
|---|---|---|---|---|
| total Tau/Ab-2 |
1.5 to 2/125 |
1.5 |
10% WBR1/1 |
1.5 |
| PHF-Tau | ||||
| pTauT205 | ||||
| APP, C-terminal |
2/125 or 2.5/1003 |
1.5 |
10% FBS2/2 |
1 |
| APP (A4) |
3/1253 |
1.5 |
10% WBR/1 |
1.5 |
| β-Actin4 | 10% WBR/1 | 1 |
FBS, fetal bovine serum (heat-inactivated); paired helical filament; WBR, Western blocking reagent (Roche Applied Science).
1Incubation of membrane in boiling PBS for 5 minutes recommended.
2Incubation of membrane in boiling PBS for 5 minutes required.
3Western blotting apparatus kept in the water bath at room temperature to prevent overheating.
4β-actin was measured for each blot after stripping (2% SDS at 56°C for 30 minutes).