INTRODUCTION
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).
This protocol was developed for the BIORAD protein gel and transfer apparatus. The buffers can be used with any electrophoresis/transfer system.
Solutions.
.Transfer Buffer
25 mM Tris 3.03 g Tris base
192 mM glycine 14.4 g glycine
20% MeOH 200 ml methanol
up to 1 liter with Q
do not adjust the pH it should be 8.3
10X TBS
100 mM Tris 8.0 100 ml 1 M Tris pH 8.0
1.5 M NaCl 87 g NaCl
up to 1 liter with Q
to make TBST add Tween 20 to 0.1%
to make prehybridization solution add serum to TBST to 5%
mix approximately 0.5 g Ponceau-S in 500 ml 1% acetic acid
(Fisher# BP103-50)
Developing Solution
100 mM Tris 9.5 15 ml 1M Tris 9.5
100 mM NaCl 3 ml 5M NaCl
5 mM MgCl2 0.75 ml 1M MgCl2
up to 150ml with Q
To use, add 66 ml NBT and 50 ml BCIP to 15 ml of Developing Solution.
NBT (sigma N-6876) 75 mg/ml in DMF, and BCIP (sigma B-6777 (pToluidine Salt) 50 mg/ml in DMF. Store at -20°C.
Resolving Gels:
Gel concentration of 12.5% in 0.25 M Tris-HCl pH 8.8
Volume Volume
Reagent: (ml: TO MAKE 30 ML) (ml: TO MAKE 10 ML)
40% Acrylamide stock*: 9.4 3.1
water (distilled) 12.3 3.8
1 M Tris-HCl pH 8.8 7.5 2.5
10% SDS 0.3 0.1
Peroxydisulphate 1% 0.5 0.5
TEMED (added last) 20 ul 20 ul
* = 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).
Stacking Gels:
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
1 M Tris-HCl pH 6.8 1.9 1.25
10% SDS 0.15 0.1
Peroxydisulphate 1% 0.5 0.5
TEMED (stir quickly) 20 ul 20 ul
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.
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.
Sample Preparation:
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).
Procedure
1.Run an electrophoretic separation of known antigenic proteins
2.Draw a line 0.5 cm from the top edge of an 8 x 10 cm nitrocellulose sheet and soak it in blot buffer for about 5 minutes.
Nitrocellulose is both fragile and flammable and easily contaminated during handling. Wear gloves which are prewashed.
When soaking the nicrocellulose wet first one side and then turn the sheet over and wet the other, to prevent trapping air within the filter.
3.Place 200 ml of blot buffer into a tray and add a piece of filter paper slightly larger than the electrophoretic gel from Step1.
4.Remove the gel from the electrophoresis chamber after the proteins have been separated, and place the gel into the tray containing the filter paper. Do not allow the gel to fall onto the paper, but place it next to the paper in the tray.
5.Gently slide the gel onto the top of the filter paper. Keep the stacking gel off of the paper until the last moment, since it tends to stick and make repositioning difficult.
6.Holding the gel and the filter paper together, carefully remove them from the tray of blot buffer and transfer the paper and gel to a pad of the blot cell with the gel facing up.
7.Transfer the nitrocellulose sheet (ink side down) onto the top of the gel and line up the line drawn on the sheet with the top of the stacking gel.
Once the gel and nitrocellulose touch they can not be separates.
8.Roll a glass rod across the surface of the nitrocellulose to remove any air bubbles and insure good contact between the gel and nitrocellulose.
9.Lay another sheet of wet filter paper on top of the nitrocellulose creating a sandwich of paper-gel- nitrocellulose-paper, all lying on the pad of the blot cell.
10.Add a second pad to the top of the sandwich and place the entire group inside of the support frame of the blot cell, and assemble the blot cell so that the nitrocellulose side of the sandwich is toward the positive terminal.
11.Check that the buffer levels are adequate and that the cooling water bath is adjusted to at least 5° C. Subject the gel to electrophoresis for 30 minutes with the electrodes in the high field-intensity position. Follow the manufacturer directions during this phase. Failure to closely monitor the electrophoresis buffer or temperature can result in a fire. Use a circulating cold bath appropriate to the apparatus and hold the voltage to a constant 100 vdc.
12.Upon completion of the electrophoresis (timed according to manufacturer's directions), turn off the power and disassemble the apparatus. Remove the blot pads from the sandwich and remove the filter paper from the nitrocellulose side.
13.Place the sandwich, nitrocellulose side down, onto a glass plate and remove the other filter paper.
14.Use a ball point pen to outline the edges of the separating gel onto the nitrocellulose, including the location of the wells.
Carefully lift the gel away from the nitrocellulose and mark the locations of the pre-stained molecular weight standards as the gel is peeled away. Peel the gel from the separating gel side, not the stacking gel.
Do not allow membrane to dry out.
15.Wash the blot (the nitrocellulose sheet) at least four times with 100 ml of PBS-Tween 20 for five minutes each on a rocking platform.
16.Cut the blot into 0.5 cm strips.
17.Inactivate sera containing positive and negative antibody controls to the antigens under examination by treating at 56 ° C for 30 minutes. Make dilutions of 1:100 and 1:1000 of the controls with PBS-Tween 20.
18.Place 3 ml of the diluted sera or controls onto a strip from Step 16 and incubate for 1 hour at room temperature while continuously rocking the sample.
19.Wash the strips four times for 5 minutes each with 10 ml quantities of PBS-Tween 20. The first wash should be done at 50° C but the last three may be done at room temperature.
20.Add 3 ml of horseradish peroxidase-labeled antiglobulin, optimally diluted in PBS-Tween and incubate at room temperature for 1 hour with continuous agitation.
21.Wash the strips four times for 5 minutes each with PBS- Tween 20 and one more time with PBS only.
22.Remove the PBS and add 5 ml of substrate solution. Positive reaction bands usually appear within 10 minutes. Stop the reaction by washing with water. Refer to Figure 4.15 for a comparison.
Notes
One of the more difficult tasks of electrophoretic separations is the identification of specific bands or spots within a developed gel. As observed with LDH isozymes, one method of doing this is to react the bands with an enzyme substrate that can be detected colorimetrically.
As a rule, however, most peptides are denatured during electrophoresis, and, of course, nucleic acids have no enzyme activity.
The methods employed for identifying non- enzymatic proteins and nucleic acids have been termed Western for immunoblotting of proteins, Southern for techniques using DNA probes Northern when using RNA probes. The probes are radioactive complimentary strands of nucleic acid. The first of these techniques was the Southern, named for the developer of the procedure, Edward Southern. Northern and then Western blots were named by analogy.
Blotting techniques first develop a primary gel: protein on acrylamide; or DNA/RNA on agarose. The gel patterns are then transferred to nitrocellulose membrane filters and immobilized within the nitrocellulose membrane. This process of transfer to an immobilizing substrate is where the term blotting originated. The process is widely used in today's laboratories because the immobilization allows for extensive biochemical and immunological binding assays that range from simple chemical composition to affinity purification of monospecific antibodies and cell- protein ligand interactions.
In practice, the electrophoresis gel is sandwhiched between two layers of filters, two foam pads (for support) and two layers of a stainless steel mesh. This entire apparatus can be submerged in a buffer and transfer allowed to occur by diffusion (yielding two blots, one on each filter), or can be arranged in an electro-convective system so that transfer occurs in a second electrophoretic field.
Once the transfer has occurred, the blots can be probed with any number of specific or non-specific entities. DNA can be probed, for example, with cDNA or even a specific messenger RNA to identify the presence of the gene for that message.