Polyacrylamide gel electrophoresis allows protein separation by differentiation migration to anode or cathode through a non-reactive matrix formed by acrylamide and N,N-methylene bisacrylamide comonomers that undergo free radical mediated polymerisation to form a meshwork of pores that sterically resist protein migration. Polymerisation is initiated by ammonium persulfate (radical source) and catalysed by TEMED (a free radical donor and acceptor).The relative molecular weight (MR) of protein separation is determined by the local acrylamide concentration (acrylamide and bisacrylamide) and the acrylamide:bisacrylamide ratio.
In the absence of SDS (Sodium dodecyl sulphate), the extent and direction of protein migration depends largely on the individual charge to mass rations; and this complicates MR estimations. SDS (an anionic detergent) partially denatures protein secondary structure and non-disulphide-linked oligomeric structures, conferring a nett negative charge on proteins due to the highly anionic sulphate group on the detergent. Addition of reducing agent (e.g. 2-mercaptoethanol or dithithreitol) and heating allows complete denaturation by breaking the intra- and inter-disulfide bridges and by linearising the polypeptide chains by thermally overcoming folding kinetics. Polypeptide linearization and SDS binding allow migration to occur solely on the basis of MR since all protein species carry nett negative charges and display an anodal migration.
For an improved separation in SDS-PAGE, discontinuous, rather than continuous buffer system is preferred (A continuous system has only a single separating gel; and uses the same buffer in the tanks and the gel). In a discontinuous buffer system, a non-restrictive large pore gel, called a stacking gel, is layered on top of a separating gel called a resolving gel. Each gel is made with a different buffer, and the tank buffers are different from gel buffers.
Laemmli discontinuous PAGE system
Electrophoretic stacking and separation of proteins by Laemmli system relies on the different glycine ionisation states (pka 1 =2.45; pKa 2 =9.6; pI=6.025) that occur between the stacking and running gels. The stacking gel usually has a low total acrylamide concentration (%T) ranging between 3-5%, which does not sterically hinder most proteins and has an approximate pH of 6.8 at 4°C. From Handerson-Hasselbach equation, 0.0015% of the glycine species are estimated to be anionic, and hence electrophoretically mobile. At pH 6.8, the rest predominate as glycine Zwitterions that carry no charge. The running conditions for Laemmli gels are constant current (i.e. constant charge per unit time) and in order to maintain this, the anionic protein species carry the charge in place of zwitterions. This allows the proteins to stack in a thin band behind the highly mobile chloride ions. Entry into the resolving gel presents the protein band with both pH increase and a pore size decrease (since %T can range from 5-20%). Deprotonation of the glycine zwitterions in the basic (pH 8.8) resolving gel allow an increase in [anionic glycinate] (from 0.005% to 15.8%). This decreases the amount of charge the proteins are required to carry and allows them to separate with respect to MR within the resolving gel.
Bio-Rad vertical slab gel apparatus for SDS-PAGE
Acrylamide/bisacrylamide monomer stock solution (30%T, 2.6%C). Acrylamide monomer (58.4 g) and N,N–methylenebisacrylamide (1.6 g) are dissolved and made up to 200 ml with dd. H2O and degassed. The solution is filtered through Whatman No. 1 filter paper and stored at room temperature in an amber bottle.
4 X Running gel buffer (1.5 M Tris-HCL, pH 8.8). 36.3 g of Tris are dissolved in 180 ml dd.H2O, titrated to pH 8.8 with HCL and made up to 200 ml. The buffer is stored at 4°C.
4 X stacking gel buffer (500 mM Tris-HCL, pH 6.8). 12 g Tris is dissolved in 180 ml dd.H2O, then titrated to pH 6.8 with HCL and made up to 200 ml. The buffer is stored at 4°C.
SDS Stock Solution [10% (w/v)]. 10 g SDS is dissolved in dd.H2O and made up to 100 ml with dd.H2O. The solution is stored at room temperature.
Ammonium persulfate initiator solution [(10% (w/v)]. 0.05 g Ammonium Persulfate is dissolved in 500µL dd.H2O just before use. The solution is kept at 4°C for up to 1 week.
5 X Electrode (running) buffer or Tank buffer [125mM Tris-HCL, 960mM glycine, 0.5% (w/v) SDS, pH 8.3]. 30 g Tris, 144 g glycine and 10 g SDS are dissolved in dd.H2O and made up to 2 litres without pH adjustment and stored at 4C. When required, 300 ml 5 X stock solution is diluted with 1.2 litres ice-cold dd. H2O (1:4 dilution).
5 X Non-reducing treatment buffer [62.5 mM Tris-HCL, pH 6.8, 20 % (v/v) glycerol, 2% (w/v SDS)]. 3.4 ml dd.H2O, 1 ml 4 X stacking gel buffer, 1.6 ml glycerol, 1.6 ml of 10% (w/v) SDS stock solution, and bromophenol [400 μL of 0.5% (w/v) solution in dd.H2O] are mixed together and stored at room temperature. When required, the sample buffer is diluted 1:4 with the protein samples and boiled for 5 min before flash cooling on ice and loading onto SDS-PAGE gels.
5 X Reducing treatment buffer [62.5mM, Tris-HCL, pH 6.8, 20% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol]. Prepared as non-reducing buffer except for addition of 0.4 ml (400 μl) 2-mercaptoethanol.
Molecular markers[phosphorylase b (94KDa), BSA (68KDa), Ovalbumin (45KDa), carbonic anhydrase (30KDa), Soybean Trypsin inhibitor (20.1 KDa), and ±-lactalbumin (14.4 KDa)]. Lyophilised markers are reconstituted in 100 ml reducing treatment buffer and boiled for 5 min. Aliquots are stored @ 4°C. pre-stained markers can be purchased from Fermenters.
- SDS-PAGE electrophoresis unit (Hoefer® Mighty Small) is assembled according to manufacturers instructions.
- Glass plates, aluminium backings, plastic combs and 1.5 mm plastic spacers are cleaned with detergent and then with 70% (v/v) ethanol. The glass plates, spacers and aluminium backings are assembled in a gel caster, the bottoms sealed and tested for leakage.
- Acrylamide monomer, dd.H2O, gel buffer and SDS are mixed with fresh ammonium persulfate and TEMED as indicated for the running gel (in Table 1), loaded into sealed gel caster (overlaid with dd.H2O to exclude oxygen) and allowed to polymerize. Isopropanol can be used to overlay the gel mixture in place of water.
- After polymerization, the dd.H2O is decanted and the stacking gel, made as described in table 1, and layered on top of the polymerized running (resolving) gel. Plastic 10 well combs are inserted into the stacking gel and polymerization allowed to occur (~30 min).
- The combs are then removed, gels assembled into electrophoresis units and pre-electrophoresed (for 30 min, ~30 mA) before sample loading, to remove excess free radicals. Alternatively, wells can be thoroughly washed with water to remove acrylamide and excess radicals instead of pre-electrophoresis.
- During electrophoresis the gel is cooled using a circulating water bath (4°C) and protein samples are separated at 35 mA constant current (for big Bio-Rad gels) until dye front enters running gel, then current is increased to 70 mA. [Small gels are run at 15 mA initially, then 30 mA constant current].The gel is either further processed (e.g. zymography or western blotting) or stained for protein.
Table 1: Reagent composition and proportions for two Tris-glycine gels
|Reagents||Resolving Gel %||Stacking Gel %|
|Resolving gel buffer (ml)||3.75||3.75||3.75|
|Stacking gel buffer (ml)||1.75||1.75|
|10% SDS (μl)||150||150||150||70||70|