What are the basic steps in two-step capillary isoelectric focusing?

Two-Step CIEF: 

1. Overcoming Electroosmotic Flow (EOF): Coat the capillaries to reduce or eliminate EOF, which can disrupt the focusing process and lead to loss of resolution and reproducibility.
2. Focusing Step: Fill the entire capillary with the sample-ampholyte mixture, and apply a high voltage. Focusing progress is monitored by the decrease in operating current until equilibrium is reached. 
3. Mobilization Step: Once focusing equilibrium is attained, mobilization begins. This may involve applying pressure, vacuum, gravity, or electrophoretic forces while maintaining the electric field. 
4. Electrophoretic Mobilization: In electrophoretic mobilization, the composition of the anolyte or catholyte is altered, causing a progressive pH shift down the capillary. This prompts electrophoretic migration of focused proteins past the detection point.
5. Detection: The entire capillary is monitored using techniques such as UV absorbance or native fluorescence to visualize the separated protein bands. This allows for tracking the focusing of specific protein species over time.