非变性胶蛋白电泳

1. Introduction

SDS-PAGE (Section 2.2) is probably the most commonly used gel electrophoretic system for analyzing proteins.

However, it should be stressed that this method separates denatured protein. Sometimes one needs to analyze native,nondenatured proteins, particularly if wanting to identify a protein in the gel by its biological activity (for example, enzyme activity, receptor binding, antibody binding, and so on). On such occasions it is necessary to use a nondenaturing system such as described in this section. For example, when purifying an enzyme, a single major band on a gel would suggest a pure enzyme. However this band could still be a contaminant; the enzyme could be present as a weaker (even nonstaining) band on the same gel. Only by showing that the major band had enzyme activity would you be convinced that this band corresponded to your enzyme. The method described here is based on the gel system first described by Davis ( 1). To enhance resolution a stacking gel can be included (see Section 2.2 for the theory behind the stacking gel system).

2. Materials

1. Stock acrylamide solution: 30 g acrylamide, 0.8 g bis-acrylamide. Make up to 100 mL in distilled water and filter. Stable at 4 ℃ for months (see Note 1). Care: Acrylamide Monomer Is a Neurotoxin. Take care in handling acrylamide (wear gloves) and avoid breathing in acrylamide dust when weighing out.

2. Separating gel buffer: 1.5M Tris-HCl, pH 8.8.

3. Stacking gel buffer: 0.5M Tris-HCl, pH 6.8.

4. 10% Ammonium persulfate in water.

5. N,N,N',N'-tetramethylethylenediamine (TEMED).

6. Sample buffer (5X). Mix the following:

a. 15.5 ml of 1M Tris-HCl pH 6.8;

b. 2.5 ml of a 1% solution of bromophenol blue;

c. 7 ml of water; and

d. 25 ml of glycerol.

Solid samples can be dissolved directly in 1X sample buffer. Samples already in solution should be diluted

accordingly with 5X sample buffer to give a solution that is 1X sample buffer. Do not use protein solutions that are in a strong buffer that is not near to pH 6.8 as it is important that the sample is at the correct pH. For these samples it will be necessa

7. Electrophoresis buffer: Dissolve 3.0 g of Tris base and 14.4 g of glycine in water and adjust the volume to 1 l. The final pH should be 8.3.

8. Protein stain: 0.25 g Coomassie brilliant blue R250 (or PAGE blue 83), 125 ml methanol, 25 ml glacial aceticacid, and 100 ml water. Dissolve the dye in the methanol component first, then add the acid and water. Dyesolubility is a problem if a different order is used. Filter the solution if you are concerned about dye solubility. Forbest results do not reuse the stain.

9. Destaining solution: 100 ml methanol, 100 ml glacial acetic acid, and 800 ml water.

10. A microsyringe for loading samples.