5.2.3 Sequential Incubation of Additional Targets on the Same Membrane

If the membrane needs to be incubated with antibodies for other targets subsequently, the target with relatively lower expression should be incubated first, followed by the one with higher expression. Meanwhile, there should preferably be a molecular weight difference of approximately 10 kDa between the two targets. Additionally, the secondary antibodies used for the two targets should be as different as possible; if this is unavoidable, the incubation can be performed on two separate membranes.
Experimental Procedures
1. TBST Washing
First, wash the ECL solution off the membrane with TBST, performing 3 washes with 5 minutes per wash.
2. Strip Buffer Treatment
After preparing the strip buffer solution according to the instructions provided with the strip buffer, wash the membrane at room temperature for an appropriate duration.
3. Exposure and Development
Incubate the membrane with the primary and secondary antibodies for the new target following the same procedure as for the previous target. Subsequently, perform exposure and development to obtain the experimental results.
4. Note:
If a labeled primary antibody is used, the secondary antibody incubation step and the subsequent membrane washing process can be skipped, and detection can be performed directly.