Capillary electrophoretic analysis of proteins

Summary

The main features of capillary electrophoresis are high column efficiency (N＞105-106), short analysis time, low consumption of sample volume and reagents, multiple modes of operation, easier to change the background electrolyte, on-line detection and automation, which make it an analytical technique complementary to HPLC, and it has shown a good application prospect in the field of life sciences. The analysis of peptides and proteins has evolved from the initial optimization of standard sample mixtures towards applications such as quantitative determination of recombinant proteins and their contaminants, CE/MS for protein characterization, CE-immunoassay, ligand-biomolecule binding studies, and enzyme activity analysis. It has been more widely used in genetic engineering, biochemistry, clinic, and analysis of natural products, and has become one of the most attractive technologies in the field of genetic engineering for monitoring recombinant protein production, downstream processing, and analysis of final products. It is used in clinical chemistry for the isolation of endogenous peptides and the diagnosis of hemoglobinopathies. Used in food analysis for milk proteins, soybean hydrolysis products, etc. Source: Handbook of Protein Technology

Operation method

basic program

Materials and Instruments

Protein Solution
Capillary Electrophoresis

Move

"Selection of operating conditions" is specified in "Others".

1. Preparation of the capillary electrophoresis column: cleaning, reaction and column equilibration;

2. removal of the buffer solution cell at the inlet end and replacement with a sample tube;

3. Inject the sample using low pressure or electromigration. 4;

4. replacing the buffer cell with another buffer cell;

5. apply the voltage required for separation. Perform electrophoretic analysis.

Common Problems

Selection of operating conditions

1. Capillary size

The most commonly used capillary tubes are fused silica capillary tubes with an inner diameter of 25-75 um. The effective length of the capillary tubes (the distance from the sample inlet end to the detector window) is commonly used to be 20-50 cm, which should be as short as possible in terms of analyzing time.

2. High Voltage Power Supply

The high voltage power supply used for HPCE should be able to provide a DC voltage of 30 kV and a current of 200-300 uA. The voltage must be stabilized at ±0.1% to achieve high reproducibility of the migration time. The polarity of the power supply should be switchable. Constant voltage is the most commonly used method, but when isokinetic electrophoresis is performed or the temperature cannot be well controlled, constant current or constant power should be used to obtain a constant migration time.

3. Temperature control

Since the feed and migration times are determined by the viscosity of the solution, the temperature should be controlled to ±0.1°C, which is important for the reproducibility of the operation. It is more efficient to use a liquid than a gas thermostat.

4. Sample Volume

The sample zone length should be less than 1% to 2% of the capillary length. Overloading of the sample will result in broadening of the peaks and distortion of the peak shape.

5. Sample Inlet Method

With the hydrodynamic method, the sample volume is basically not affected by the sample matrix; for electromigration injection, the sample volume is determined by the electrophoretic mobility of each component, and the influence of discrimination effect should be noted.

6. Sample preconcentration

In isochronous electrophoresis and isoelectric focusing analysis, the sample can be concentrated many times in the steady state, which is favorable for UV detection and analysis. However, in capillary zone electrophoresis, the sample zone will be broadened, so it is often necessary to use sample stacking, isochronous electrophoresis and chromatography to achieve the purpose of sample stacking through the electrodynamic injection of low-conductivity media or pressure injection. Discontinuous buffer systems are used for on-column concentration of low-concentration samples with injection volumes up to 30 times larger than conventional methods. With whole capillary injection techniques, samples can be concentrated 400 to 1000 times.

Transient isotachophoretic concentration can be achieved either with a canonical isotachophoretic electrolyte system or by supplementing the sample with a high-flux coion of the same charge sign as the separated sample, while the electrolyte's coion has a low-flux. During electrophoresis, there is a gradual change from an isotropic to a zonal electrophoretic mode.

Chromatographic preconcentration involves trapping the sample in a reversed-phase or affinity packing material and then eluting it with a small amount of solvent while the sample is purified, such as protein G and stationary phases incorporating iminodiacetic acid metal chelating functional groups. Concentration using a very low concentration of buffer and a polyacrylamide gel with a very small pore size, where the water in the sample diffuses into the gel and the salts are simultaneously removed, can be applied to small amounts of sample.

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