2. Materials and methods
2.24. Sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
The SDS-PAGE method is based on the method of Laemmli (1970). SDS is an anionic detergent that has two effects on proteins: first, it leads to disbanding of oligomeric structures, resulting in a relaxed conformation and, second, it binds in excess in a constant relation (1.4 g SDS/1 g protein) to the protein, which leads to an equal negative charge per mass unit. Therefore, in the presence of SDS, proteins can be separated based on their molecular weight.
The negatively charged SDS-protein complexes run towards the anode in an electrical field. To separate these complexes, the molecular-sieve properties of polyacrylamide gel (PAA-Gel) are used, where the mobility of most proteins is reversely proportional to the decimal logarithm of their molecular weight. The discontinuous buffer system of Laemmli (1970) leads to the concentration of probes on the transition state between a stacking gel and a separation gel, resulting in a higher resolution of the following separation of proteins. To determine the molecular weight of unknown proteins, a marker containing some proteins with molecular weights of 14-97 kDa („Low Molecular Weight Calibration Kit“; Amersham Pharmacia, Freiburg, Germany) or 9 to 185 kDa (“Bench Mark Prestained Protein Ladder”, Invitrogen, Groningen, Netherlands) was loaded on the same gel. The pore size of a PAA-gel depends on relation between the concentrations of acrylamide to cross-linking N-N´-methylenbisacrylamide. The acrylamide quota and the cross-linking degree are the values that characterize a polyacrylamide gel. To separate total protein extracts and detect sucrose synthase (SuSy), consisting of 92- to 93-kDa subunits (Hohnjec et al., 1999), a PAA-gel containing 6 or 7.5 % polyacrylamide in the separating gel and 4 % acrylamide in the stacking gel were prepared. The separating range was 60 to 200 kDa in 6 % and 40 to 200 kDa in 7.5
% PAA-gel. Gel plates, spacer and combs as well as gel camera were ordered from Biometra (Göttingen, Germany).
2.24.1. Buffers and solutions for analytic SDS-PAGE
Acrylamide / Bisacrylamide 29.2 % (w/v) acrylamide
(30 % T/2.7 % C) stock solution (30%) 0.8 % (w/v) N,N´-methylene bisacrylamide Dissolve in 100 ml dd H2O and filter through 3 MM paper
Store at 4-10 oC
4 × Stacking gel buffer 500 mM Tris-HCl pH 6.8 0.4 % (w/v) SDS
4 × Separating gel buffer 1.5 M Tris-HCl pH 8.8 0.4 % (w/v) SDS
10 % Ammonium persulfate 10 % (w/v) Ammonium persulfate (in dd H2O) (APS) solution stored at 10 oC
10 × SDS Electrophoresis buffer 250 mM Tris
1.92 M Glycine
1 % (w/v) SDS
pH 8.2-8.4 (not adjusted)
5 × Sample buffer 60 mM Tris-HCl (pH 6.8) 2 % (v/v) β-Mercaptoethanol
25 % (v/v) Glycerol
2 % (w/v) SDS
0.1 % (v/v) Bromphenol blue
2.24.2. Pouring the SDS-Polyacrylamide gel
Glass gel plates were cleaned with acetone or ethanol, silicon spacer tubes and combs were cleaned with water. The glass plates were joined together to form the cassette and clamped in vertical position. Solutions for stacking and separating gel were mixed as described in the table below. To start the polymerisation, TEMED (N,N,N‘,N‘-Tetramethylene diamine) solution was added to the separating gel solution. The gel solution was mixed and poured into the gel chamber up to 1 cm under the ends of the
comb slots. To ensure that the gel polymerized with a smooth surface, a 2 mm layer of dd H2O was carefully poured on the gel using a Pasteur pipette. Because of the great difference in density between dd H2O and the gel solution, the water is spread across the surface of the gel without much mixing. When the gel was set, a clear refractive index change could be seen between the polymerised gel and the overlaying water. Then the overlaying water was poured off and TEMED was added to the stacking gel solution to start polymerisation. The stacking gel was poured into the gel cassette until the solution reached the upper edge and the slot-forming comb was put into the solution before leaving the gel to set. 30 min to 1 hour later, the comb was removed from the gel, and the gel slots were rinsed with electrophoresis buffer to remove any rests of non-polymerised acrylamide solution.
The data of the following table are based on the stock solutions and buffers given in 2.24.1. The volume is sufficient for two mini gels (8.5 cm × 7.5 cm).
Separating gel, 7.5 % (15 ml): Acrylamide stock solution (30 %) 3.750 ml 4 × Separating gel buffer 3.750 ml
dd H2O 7.410 ml
10 % APS solution 75 µl TEMED (add before pouring the gel) 15 µl
Stacking gel, 4 % (5 ml): Acrylamide stock solution (30 %) 0.670 ml 4 × Stacking gel buffer 1.250 ml
dd H2O 3.050 ml
10 % APS solution 30 µl TEMED (add before pouring the gel) 5 µl
The resulting end concentrations were:
Stacking gel Separating gel
Tris/HCl pH 6.8 0.13 M - Tris/HCl pH 8.8 - 0.38 M SDS 0.1 % (w/v) 0.1 % (w/v) Acrylamide 4.6 % (w/v) 7.5 % (w/v) N,N-Methylene bisacrylamide 0.12 % (w/v) 0.33 % (w/v) APS 0.06 % (w/v) 0.05 % (w/v)
2.24.3. Probe preparation and SDS polyacrylamide gel electrophoresis (SDS-PAGE) Ca. 15 µg protein per slot were used for immunoblotting and 10-30 µg protein for Coomassie stained gels.
The samples were supplemented with sample buffer to a final concentration of ≥ 1× sample buffer to ensure proper solubilisation of the proteins and solubilised for 15 min at RT by sucking the solution up and down with a pipette. Then the samples were boiled at 95 °C for 3 min and centrifuged afterwards at top speed (13 000 rpm) for 5 min in a table top microcentrifuge to sediment insoluble material. After removing the spacers, the gel cassette was assembled with the electrophoresis unit, and first the top and then the bottom reservoir were filled with 1× SDS electrophoresis buffer (see 2.24.1.). Then the appropriate volume of sample was filled up to 10 µl with sample buffer (see 2.24.1.) and loaded into the SDS gel slot using a microsyringe. Every empty slot on the gel was filled in with 10 µl sample buffer. The electrophoresis was run under a constant current of 25 mA (500 V, 100 W) and was continued until the Bromphenol blue reached the bottom of the gel (about 1.5 hours). Then the gel cassette was removed from the electrophoresis unit and opened, and the gel was taken out to stain in Coomassie or to mount a Western blot.
2.24.4. Coomassie staining of SDS polyacrylamide gels
Coomassie staining serves to detect proteins in an SDS polyacrylamide gel. For this purpose, the gel was fixed after electrophoresis and stained in staining solution (see below) for at least 30 min and then destained in wash solution for about 1-2 hours until the gel background was colourless. After washing, the gel could be stored for a long time in dd H2O. Alternatively, it could first be soaked in 10 % glycerol for 30 min and then dried under vacuum.
Coomassie staining solution 0.25 % (w/v) Coomassie Brilliant Blue R 250 40 % (v/v) Methanol
10 % (v/v) Acetic acid
Washing solution 50 % (v/v) Methanol
10 % (v/v) Acetic acid