1. Place the membrane in the primary antibody solution and incubate with agitation for 1 h at room temperature or overnight at 4°C.
2. Place the membrane in washing solution and wash three times for 5 min per wash.
3. Place the membrane in the biotinylated secondary antibody solution and incubate with agitation for 1 h at room temperature or 37°C.
4. Place the membrane in washing solution and wash three times for 5 min per wash.
5. Dilute the streptavidin-HRP conjugate (or streptavidin CyDye conjugate if using Amersham ECL Plex detection) in PBS-Tween or TBS-Tween.
6. Incubate the membrane in the solution for 45 to 60 min at room temperature on an orbital shaker.
7. Briefly rinse the membrane with two changes of washing solution.
8. Wash the membrane by suspending it in enough washing solution to cover the membrane and agitate for 5 min at room temperature. Repeat the procedure at least four to six times.
9. Continue with detection as recommended for the selected detection reagent and imaging system.
여기서 11. 7.2.2입니다.
왜 3layer 라고 명칭이 붙여진지도 모르겠고
다른 probing 단계와의 차이점을 잘 모르겠습니다...
#western blotting #probing #3 layer probing