Introduces a capillary electrophoresis technology that can be used for protein and peptide separation
This document provides a technical guide for performing capillary isoelectric focusing (cIEF) using an Agilent capillary electrophoresis system. The method allows for the separation of proteins and peptides based on their isoelectric points (pI), which is useful for identification, purity assessment, and clinical diagnosis.
The protocol details a two-step process: first, focusing proteins into sharp zones within a pH gradient formed by ampholytes, and second, mobilizing the contents past the detector. It includes specific instructions on sample preparation to prevent precipitation, such as adding Triton X-100 and Hydroxyethylcellulose (HEC), along with detailed recipes for preparing reagents like the ampholyte mixture, catholyte, and anolyte.
We recommend using a protein concentration range from 0.5 to 2.5 mg/mL.
The supplied 64.5 cm x 50 µm i.d. capillary should be cut to a total length of 48.5 cm, providing an effective length of 40 cm.
Anolyte is position 4, Catholyte is position 5, Acidic wash is position 3, Blank mix is position 2, and Waste (100-µL water) is position 1.