Table 2. Western blot protocol for the identification of the epitopes recognized by anti-α-SKA (clone 10D2) and anti-α-CAA (clone 22D3).
| Protocol steps | Reagent | Concentration/
dilution |
Time |
|---|---|---|---|
| Migration of Purified α-SKA and α-CAA on
SDS-PAGE |
1μg/lane | overnight | |
| • Transfer on Nitrocellulose membranes.
• Membranes cut in strips for Ab-peptides incubation. |
2 h | ||
| Preincubation of antibodies with peptides | MAb:
• anti-α-SKA (10D2) • anti-α-CAA (22D3) For details, see Table 1 Blocking Peptides (see Figure 1) |
1:5000 1:5000 100 μg/ml |
1 h |
| Membranes pretreatment | 5% dried skimmed milk in TBS | 5% | 1 h |
| Membranes Incubation with antibody-peptides | In 0.1% TX-100/TBS | 2 h | |
| Membranes Incubation with 2 nd antibody | Goat Anti-Mouse IgG (H+L) HRP
Conjugate (BioRad) #1706516 In 0.1% TX-100/TBS |
1:10’000 | 1 h |
| HRP activity development | ECL protocol according to (RPN
2109; GE Healthcare), |
5–60 sec |