This is a transfer sandwich, and all parts must be aligned carefully for accurate transfer of protein, also, it is recommended that all air bubbles be removed by gently rolling with a glass rod as each layer of the sandwich is added. Additional sandwiches may be added on top of the original but each complete sandwich should be separated from the next by a piece of PVDF membrane to prevent movement of protein from one sandwich to the next. When gel sandwiches are complete replace the cathode plate and the cover on the blotter. Connect the power supply and turn on. The second half will be probed with antibodies.
Antibody probing is done in the following manner.
Next you wash the gel with buffer 3 times for 5 minutes. Wash three more times and label the membrane by adding the substrate for the conjugate as per instructions. When adequate labeling is apparent, rinse the membrane with distilled water to remove additional substrate and stop development of additional color.