CE is particularly useful for separating specific peptides from complex mixtures such as proteolytic products. The following protocol describes the use of the P/ACE 5000 CE instrument (Beckman) with low pressure sample injection. However, other instruments can be used to perform the same separation according to the manufacturer's instructions. Source: Compact Laboratory Guide to Molecular Biology (5th Edition)
Operation method
basic program
Materials and Instruments
Peptide Mixture Move 1. Pretreat the capillary by flushing with the following solution: 10 times the column volume of 0.1 mol/L NaOH at low pressure (0.5 lb/in2 ) 10x column volume of water 4x column volume of 0.25 mol/L sodium phosphate buffer, pH 2.30. Store the column in 0.25 mol/L sodium phosphate buffer, pH 2.30, at 25 °C. 2. 2. Prepare the peptide mixture by dissolving 10 nmol (approximately 300 g) in 10 ml of 0.05 mol/L sodium phosphate buffer, pH 2.30. Freeze the unused mixture at 100 μl per tube. 3. 3. Sample 10-20 nl of sample at 0.5 lb/in2 in 10 s by low-pressure injection. 4. 4. Separate the peptide mixture using the following conditions: Electrolyte: 0.05 mol/L sodium phosphate buffer, pH 2.3 Detection wavelength: 200 nm Temperature: 25°C Voltage: 25 kV 5. Wash the column with the following solution: 0.5 min, water 1.0 min, 0.1 mol/L NaOH 1.5 min, water 1.0 min, 0.25 mol/L sodium phosphate buffer, pH 2.30 Store columns at room temperature in running buffer or water. For more product details, please visit Aladdin Scientific website.
0.05 mol/L and 0.25 mol/L sodium phosphate buffer (pH 2.30 stored at 4°C) 0.1 mol/L sodium hydroxide
75 μm I.D. fused silica capillary column CE instrumentation