Western Blot Procedure specific for ABCE1 Antibody (NB400-116)

Western Blot Procedure

1. Run 20 ul of immunoprecipitated protein on a 10% SDS polyacrylamide gel
2. Transfer protein to Immobilon-P nylon membrane: 60mA for 2 hours
3. Block membrane with 5% non-fat milk in PBS + 0.4% Tween-20 [PBS-T] for 1 hour @ RT or overnight @ 4C
4. Wash membrane with PBS-T, 3x [15 min., 5 min., 5 min.], shaking gently
5. Incubate membrane with 1:500 dilution of anti-ABCE1 (NB 400-116), diluted in PBS-T, for 1 hour @ RT
6. Wash membrane with PBS-T, 3x [15 min., 5 min., 5 min.], shaking gently
7. Incubate membrane with anti-rabbit IgG-HRP secondary, diluted in PBS-T, for 1 hour @ RT
8. Wash membrane with more than 100 ml of PBS-T at least 3x (for an 8x6 cm blot)
9. Detect cross-reacting proteins using a Chemiluminescence Reagent kit: expose ~5 seconds

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.