3: Gel Electrophoresis of Protein

I. Introduction

Electrophoresis

Biomolecules often carry positive or negative electrical charges. When placed in an electric field, charged molecules move towards the electrode of opposite charge due to the phenomenon of electrostatic attraction. Electrophoresis is the separation of charged molecules in an applied electric field. The relative mobility of individual molecules depends on several factors the most important of which are net charge, charge/mass ratio, molecular shape and the temperature, porosity and viscosity of the matrix through which the molecule migrates.

Polyacrylamide Gel Electrophoresis (PAGE)

Electrophoresis in acrylamide gels is frequently referred to as PAGE, being an abbreviation for polyacrylamide gel electrophoresis. Cross-linked polyacrylamide gels are formed from the polymerization of acrylamide monomer in the presence of smaller amounts of N,N’-methylenebisacrylamide (normally referred to as ‘bis’-acrylamide). Note that bis-acrylamide is essentially two acrylamide molecules linked by a methylene group, and is used as a cross-linking agent. Acrylamide monomer is polymerized in a head-to-tail fashion into long chains and occasionally a bis-acrylamide molecule is built into the growing chain, thus introducing a second site for chain extension. Proceeding in this way a cross-linked matrix of fairly well-defined structure is formed (Fig 5-1). The polymerization of acrylamide is initiated by the addition of ammonium persulphate (APS) and catalyzed by N,N,N’N’-tetramethylenediamine (TEMED).

Acrylamide gels are defined in terms of the total percentage of acrylamide (T %) present, and the pore size in the gel can be varied by changing the concentrations of both acrylamide and bis-acrylamide. The low percentage gels (e.g. 4%) have large pore sizes and are often used in the stacking gel system of PAGE or to separate DNA. Gels of between 9% and 20% acrylamide are often used in SDS-PAGE to separate protein according to their size.

T % = (a + b)*100/total volume (ml)

a = weight of acrylamide in g

b = weight of bis-acrylamide in g

SDS-PAGE

Proteins can be dissociated into their constitute polypeptide chains by an anionic detergent, sodium dodecyl sulphate (SDS), after the reduction of any disulphide bonds. The SDS binds to the polypeptide chain producing a rod-shaped complex, the length of which is dependent upon the relative molecular mass of the protein. The large number of these strongly anionic detergent molecules bound by the protein (approximately one SDS per two amino acids) effectively masks the native charge of the protein and at a neutral pH results in a relatively constant charge to mass ratio for all proteins. As a result, the electrophoretic mobiltity of all protein-SDS complexes is approximately equal but the molecular sieving effect of polyacrylamide gel results in a relative mobility which is inversely related to the size of the complex. Under certain conditions, this inverse relationship can be demonstrated by a linear plot of the relative mobility of the protein against the logarithm of its relative molecular mass (Fig 5-2). It is necessary to use a series of known proteins in order to produce a calibration curve.

Fig 5-2. The electrophoretic mobility of a protein on an SDS-PAGE is related to its molecular weight. A plot of log Mr of the marker protein versus relative protein mobility is linear. Adopted from Lehninger, Principle of Biochemistry, 3^rd ed.

Electrophoretic techniques

The equipment required for electrophoresis consists basically of two items, a power pack (power supply) and an electrophoresis unit. Electrophoresis units are available for running either vertical or horizontal gel systems. Vertical slab gel units of the type shown in Fig. 5-3 are commercially available and routinely used to separate protein in acrylamide gels. The gel is formed between two glass plates that are clamped together but held apart by plastic spacers. Gel dimensions are typically 12 cm x 14 cm, with a thickness of 0.5 to 1 mm. A plastic comb is placed in the gel solution and is removed after polymerization to provide loading wells for samples.

When the apparatus is assembled, the lower electrophoresis tank buffer surrounds the gel plates and affords some cooling of the gel plates. The power pack supplies a direct current between the electrodes in the electrophoresis unit. All electrophoresis is carried out in an appropriate buffer to maintain a constant state of ionization of the molecules being separated. Any variation in pH would alter the overall charge and hence the mobilities (rate of migration in the applied field) of the molecules being separated.

Sample preparation

Samples to be run on SDS PAGE are firstly boiled for 5 min in sample buffer containing b-mercaptoethanol and SDS. The mercaptoethanol reduces any disulphide bridges present that are holding together the protein tertiary structure, and the SDS binds strongly to, and denatures, the protein. It is then heated at 100℃ for 2-5 min in order to denature the protein and expose the total length of the polypeptide chian to the detergent. The sample buffer also contains a tracking dye, usually bromophenol blue, that allows the electrophoretic run to be monitored, and glycerol, which gives the sample solution density thus allowing the sample to settle easily to the bottom when injected into the loading well. Once the samples are all loaded, a current is passed through the gel and electrophoresis is performed on polyacrylamide gel and the bands subsequently visualized using an appropriate dye.

II. Materials and Methods

Stock solutions:

A. Acrylamide stock solution (30%):

29.2 g acrylamide

0.8 g bis-acrylamide

add d.H₂O to make 100 ml

B. 4X Running Gel Buffer: 1.5 M Tris-HCl, pH 8.8

C. 4X Stacking Gel Buffer: 0.5 M Tris-HCl, pH 6.8

D. Tank Buffer: 0.025 M Tris-HCl, 0.192 M glycine, 0.1 % SDS, pH 8.3

E. 5X Sample Buffer:

0.375 M Tris-HCl, 10% SDS, 50% glycerol, 12.5% b-mercaptoethanol, 0.02% bromophenol blue, pH 6.8

F. Staining solution:

0.1% Coomassie blue R-250, 10% acetic acid, 40% methanol.

G. Destaining solution: 10% acetic acid, 20% methanol.

Procedure:

i. Casting the running gel

1. Assemble gel sandwich (Fig 5-4). Add H₂O to test for leakage.

請依照圖5-4組裝電泳玻片，組裝好加入純水，看看會不會滲漏。

2. Combine solution in a flask or beaker according to Table 5-1. Acrylamide is a neurotoxin, gloves should be worn at all times. Add ammonium persulfate and TEMED last, and mix by swirling container gently.

依照下表5-1配製電泳凝膠溶液[running gel]，請注意丙烯醯胺[acrylamide]具有神經毒性，務必全程戴上手套操作實驗！過硫酸銨[ammonium persulfate]與TEMED最後再加入，輕輕晃動，將溶液混合均勻。

3. Carefully introduce solution into gel sandwich by pipetting along a spacer. This minimizes the possibility of air bubbles becoming trapped within the gel.

將電泳凝膠溶液慢慢地加入兩片電泳玻片間，不要讓氣泡產生。

4. On top of the running gel, gently layer about 1-5 mm of water to keep the gel surface flat.

為確保電泳凝膠凝固後表面的平整，在電泳凝膠溶液的上方，輕輕地加入1-5 mm高度的水，加完水後，應看到水與凝膠溶液間的介面形成一條平整的直線。

5. Allow gel to polymerize (15-30 min). When the gel has polymerized, a distinct interface will appear between the separating gel and the water, and the gel mold can be tilted to verify polymerization.

等待膠體聚合需15-30分鐘，膠體聚合好後，水與凝膠溶液間的介面會因折射度的改變，而變得更清楚。

Table 5-1 Recipes for acrylamide gel

III. Results:

I. Identify the protein bands on the gel and measure the distance it traveled in the running gel. Plot the log [MW of the markers] against the mobility for standard curve and determine their linear relationship.

II. Identify the protein in the unknown sample and estimate their molecular weight.