Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
SDS POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE)
Ed Rybicki and Maud Purves
Copyright Ed Rybicki 1996
- Support Matrices
- Separation of Proteins and Nucleic Acids
- SDS-PAGE of Proteins
- Assembling Gel Apparatus
- Electrophoresis buffer
- Sample Preparation:
- Staining of Gels:
Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the nett charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique.
Generally the sample is run in a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel. The matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run: at the end of the run, the matrix can be stained and used for scanning, autoradiography or storage.
In addition, the most commonly used support matrices - agarose and polyacrylamide - provide a means of separating molecules by size, in that they are porous gels. A porous gel may act as a sieve by retarding, or in some cases completely obstructing, the movement of large macromolecules while allowing smaller molecules to migrate freely. Because dilute agarose gels are generally more rigid and easy to handle than polyacrylamide of the same concentration, agarose is used to separate larger macromolecules such as nucleic acids, large proteins and protein complexes. Polyacrylamide, which is easy to handle and to make at higher concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore size for retardation.
Separation of Proteins and Nucleic Acids
Proteins are amphoteric compounds; their nett charge therefore is determined by the pH of the medium in which they are suspended. In a solution with a pH above its isoelectric point, a protein has a nett negative charge and migrates towards the anode in an electrical field. Below its isoelectric point, the protein is positively charged and migrates towards the cathode. The nett charge carried by a protein is in addition independent of its size - ie: the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs from protein to protein. At a given pH therefore, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules.
Nucleic acids however, remain negative at any pH used for electrophoresis and in addition carry a fixed negative charge per unit length of molecule, provided by the PO4 group of each nucleotide of the the nucleic acid. Electrophoretic separation of nucleic acids therefore is strictly according to size.
SDS-PAGE of Proteins
Separation of Proteins under Denaturing conditions
Sodium dodecyl sulphate (SDS) is an anionic detergent which denatures proteins by "wrapping around" the polypeptide backbone - and SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length - ie: the denatured polypeptides become "rods" of negative charge cloud with equal charge or charge densities per unit length. It is usually necessary to reduce disulphide bridges in proteins before they adopt the random-coil configuration necessary for separation by size: this is done with 2- mercaptoethanol or dithiothreitol . In denaturing SDS-PAGE separations therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight.
Determination of Molecular Weight
This is done by SDS-PAGE of proteins - or PAGE or agarose gel electrophoresis of nucleic acids - of known molecular weight along with the protein or nucleic acid to be characterised. A linear relationship exists between the logarithm of the molecular weight of an SDS-denatured polypeptide, or native nucleic acid, and its Rf. The Rf is calculated as the ratio of the distance migrated by the molecule to that migrated by a marker dye-front . A simple way of determining relative molecular weight by electrophoresis (Mr) is to plot a standard curve of distance migrated vs. log10MW for known samples, and read off the logMr of the sample after measuring distance migrated on the same gel.
Continuous and Discontinuous Buffer Systems
There are two types of buffer systems in electrophoresis, continuous and discontinuous . A continuous system has only a single separating gel and uses the same buffer in the tanks and the gel. In a discontinuous 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 the gel buffers. The resolution obtained in a discontinuous system is much greater than that obtained with a continuous system (read about this in any textbook).
NB: ACRYLAMIDE MONOMER IS A POTENT CUMULATIVE NEUROTOXIN. DO NOT MOUTH PIPETTE ACRYLAMIDE SOLUTIONS, AND WEAR GLOVES WHEN HANDLING UNPOLYMERISED SOLUTIONS.
Assembling gel apparatus:
Assemble two glass plates (one notched) with two side spacers, clamps, grease, etc. as shown by demonstrators or instructions. Stand assembly upright using clamps as supports, on glass plate. Pour some pre-heated 1% agarose onto glass plate, place assembly in pool of agarose: this seals the bottom of the assembly.
Gel concentration of 12.5% in 0.25 M Tris-HCl pH 8.8
Volume Volume Reagent: (ml: TO MAKE 30ML) (ml: TO MAKE 10ML) 40% Acrylamide stock*: 9.4 3.1 water (distilled) 12.3 3.8 1M Tris-HCl pH 8.8 7.5 2.5 10% SDS 0.3 0.1 Peroxydisulphate 1% 0.5 0.5 TEMED (added last) 20ul 20ul
* = 19:1 - 38:1 w:w ratio of acrylamide to N,N'-methylene bis-acrylamide
Mix ingredients GENTLY! in the order shown above, ensuring no air bubbles form. Pour into glass plate assembly CAREFULLY. Overlay gel with isopropanol to ensure a flat surface and to exclude air. Wash off isopropanol with water after gel has set (+15 min).
Gel concentration of 4.5% in 0.125 M Tris-HCl pH 6.8
Volume Volume Reagent: (ml TO MAKE 15 ML) (ml TO MAKE 10 ML) 40% Acrylamide stock 1.7 1.1 water 10.8 7.1 1M Tris-HCl pH 6.8 1.9 1.25 10% SDS 0.15 0.1 Peroxydisulphate 1% 0.5 0.5 TEMED (stir quickly) 20ul 20ul
Mix as before, then pour onto top of set resolving gel, insert comb, allow to set, remove comb, fill with electrophoresis buffer. Assemble top tank onto glass plate assembly. Fill with electrophoresis buffer.
The final TANK buffer composition is 196mM glycine / 0.1% SDS / 50mM Tris-HCl pH 8.3 , made by diluting a 10x stock solution. This goes in both top and bottom tanks.
Grind a little leaf material (eg. 2 grams) in a mortar. Centrifuge in an Eppendorf tube for 3 min. Take supernatant and mix 100ul 1:1 (v:v) with SDS-PAGE disruption mix : this is 125mM Tris-HCl pH 6.8 / 10% 2-mercaptoethanol / 10% SDS / 10% glycerol , containing a little bromophenol blue. BE CAREFUL WITH THIS AS IT SMELLS AWFUL and is poisonous to boot!!
For liquid / purified samples, take eg. 100 ul and add 50 - 100 ul of disruption mix.
Heat sample Eppendorfs for 5 min at 95oC in a "float" in a waterbath. Layer samples under buffer on stacking gels. Connect up apparatus and electrophorese as shown.
Staining of Gels:
1. Coomassie Brilliant Blue/Page-Blue 83
Make up stain: 0.2% CBB in 45:45:10 % methanol:water:acetic acid. Cover gel with staining solution, seal in plastic box and leave overnight on shaker (RT) or for 2 to 3 hours at 37 c also with agitation. Destain with 25% 65% 10% methanol water acetic acid mix, with agitation.
2. Copper Chloride (0.3M CuCl2
Rinse gel in distilled water, immerse in copper chloride solution with agitation for about 20 minutes (RT), rinse with distilled water and immerse in sufficient fresh distilled water to cover the gel (this acts as the destaining step). Seal in a plastic box (see Western Blotting for full details).