What are the steps involved in capillary isoelectric focusing?

Single-Step CIEF: 

1. Sample Introduction: Inject a mixture of sample proteins and carrier ampholytes into the capillary as a single plug at the inlet. The capillary is then placed between two electrolyte tanks: one acidic (anolyte) and one basic (catholyte). These tanks create an electric field when a voltage is applied. 
2. Simultaneous Application of Forces: Apply the electric field and a mobilizing force (like pressure, vacuum, gravity, or electroosmosis) simultaneously. 
3. Formation of pH Gradient and Focusing: As the mixture travels from the inlet to the detection point, a pH gradient forms, and proteins focus at their respective isoelectric points (pI). They focus in a narrow band due to their neutral charge at that pH.
4. 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.

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.