Analyzing peptide separation experiments

Summary

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
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

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.

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