5.2.1 Antibody Incubation

Experimental Procedures
1. TBST Washing
Wash the blocked membrane with 15 mL of TBST for 3 times, 5 minutes each time.
2. Membrane Cutting
If the membrane needs to be cut into sections according to different target proteins:
After washing the membrane, sandwich it between two pieces of transparent and clean plastic films. Cut the membrane at appropriate positions as indicated by the protein marker.
Throughout the experiment, ensure the membrane remains moist to prevent high background caused by membrane drying. Place the cut membrane sections into different compartments of the incubation box and mark them clearly.
3. Overnight Incubation
Add 10 mL of diluted primary antibody, and incubate the membrane overnight at 4°C on a shaker.
4. TBST Washing
Wash the membrane with 15 mL of TBST for 3 times, 5 minutes each time.
5. Shaker Incubation
Add 10 mL of diluted secondary antibody, and incubate the membrane at room temperature on a shaker for 1 hour.
If a fluorescent secondary antibody is used, wrap the incubation box with aluminum foil during the antibody incubation and subsequent membrane washing steps to avoid fluorescence quenching.
6. TBST Washing
Wash the membrane with 15 mL of TBST for 3 times, 5 minutes each time.