APPCter-C17 rabbit polyclonal antiserum
Antibody directed against the Cterminal
Protocol:
Protein are separated on 16.5% Tris-Tricine gels following the procedure described by Schägger and Von Jagow . Protein are transferred on 0.45 µm nitrocellulose membrane (Amersham-Pharmacia Biotech) at 2.5 mA/cm2 per gel using the semi-dry Novablot transfer system (Pharmacia Amersham Biotech).
The membrane is blocked in TBS-Tween 20 0.1% added with 5% skimmed milk during half an hour. APPCter-C17 antiserum is diluted in TBS-Tween 20 0.1% added with 4% skimmed milk at a final dilution of 1/2000. The membrane is incubated during 2 hours at ambient temperature during 2 hours or overnight at 4°C.
The incubation is followed by 3 washes in TBS-Tween 20 0.1%, 10 min each.
The secondary rabbit-IgG antibody coupled with horseradish perodixase (SIGMA Immunochemicals) is used at the final dilution of 1/4000 in TBS-Tween 20 0.1% added with 4% skimmed milk for 1 hour at ambient temperature and followed by 3 washes in TBS-Tween 20 0.1%, 10 min each.
Immunoreactive complexes are revealed with ECL™ (Amersham-Pharmacia Biotech) following the procedure of the manufacturers. A primary exposure of 15 min is necessary to to fully visualize a signal.