What are the key steps in getting good results?
The first key step in getting good results is protein extraction. First, make sure to vortex the beads with cells for 30 seconds every 10 minutes for 1 hours. This process will fully disrupt the cells/tissues and release protein. Secondly, it is very important that the cell lysate supernatant is very clear. Impurity in the supernatant ( dirty lysate ) can cause low labeling efficiency and non-specific binding. Be sure to only use the top, clear layer of the lysate supernatant for labeling. The supernatant should be as clear and transparent as water. If the supernatant still appears cloudy (unclear) at the end of the extraction protocol, freeze it at -70 for 10min, then spin at 10000xg. Save the clear layer of the supernatant.
