NARRATOR: There are a number of different commercially available systems for SDS-PAGE. Most widely used are so-called mini gels, which once they are set up and the samples are loaded, can be run in less than one hour and give adequate resolution for most purposes. Such a system will be used for the purposes of this demonstration.
There are two glass plates, one shorter than the other. In the system being used here, the taller plate has raised strips on either side which act as spacers and which will determine the thickness of the gel. In this case, the gel will be 1.5 millimetres thick.
The plates are clamped together, ensuring that they are lined up with each other, and then fixed in a casting stand. Notice how the rubber gasket under the gel plates effectively forms a seal with the bottom of the plate. The separating gel is prepared first. A mixture is prepared according to the desired percentage of acrylamide. As well as acrylamide, this mixture contains bisacrylamide for cross linking, tris buffer at pH 8.8, SDS, and reagents to catalyse the polymerisation reaction.
The separating gel mixture is poured between the previously-assembled plates. This must be done quickly before polymerisation can occur. Notice that the separating gel does not reach to the top of the plates. Space is left for addition of the stacking gel, which will be added once the separating gel is set.
The separating gel mixture is quickly overlaid with water or water-saturated butanol to exclude air from the gel and to give a smooth surface. The gel is then left to polymerise. The time required for polymerisation varies depending on the type and thickness of the gel. In this case, it is complete after about 30 minutes.
If we tilt the apparatus, we can clearly see that the gel has set. The liquid on top of the gel is the overlay. The overlay is poured away. A stacking gel mixture is now added to the top of the separating gel. This mixture is prepared in exactly the same way as the separating gel, except that it contains a lower concentration of acrylamide and is buffered at pH 6.8.
To form the wells for the protein samples, a comb of the same thickness as the gel is inserted into the stacking gel. Notice how air is excluded and the gel mixture fills the gaps between the teeth of the comb. As for the separating gel, the stacking gel is now left to polymerise.
Once the stacking gel has polymerised, the comb is removed. The bubbles here are caused by the SDS in the gel mixture. The gel plates are removed from the stand. Notice the regular wells that have been formed by the comb.
The gels, still sandwiched between the plates, are now clamped into the apparatus containing the two electrodes. The shorter plate is turned towards the centre of the apparatus and fits snugly against the rubber gasket. When the two gels are positioned in this way and the whole assembly is clamped, a watertight seal is formed around the edges of the plates.
A tris glycine running buffer containing SDS is added to the gel tank. The gel assembly is then placed in the tank, and more of the same running buffer is added to the central reservoir formed between the plates. The buffer should cover the top of the gels and fill the wells. In the tank, the buffer should reach above the bottom edge of the gel.
Notice how this apparatus effectively create two reservoirs for the running buffer. The electrodes are thin, platinum wires. The cathode is in the central reservoir, whilst the anode is bathed in the running buffer in the bottom of the tank. The only route for passage of ions between the electrodes is through the gel.
The samples to be run on the gel have been prepared by addition of a special loading buffer. This buffer contains SDS, a reducing agent, such as beta mercaptoethanol or dithiothreitol to break disulfide bridges, a blue dye, and either glycerol or sucrose to increase the density of the mixture. The samples are heated at 95 degrees C for five minutes to denature the proteins. The same samples could be run under non-reducing conditions to preserve the disulfide bridges.
The samples are loaded into the appropriate wells using a micropipette. The glycerol, or sucrose content, increases the density of the sample, and it sinks to the bottom of the well, displacing the running buffer. A mixture of molecular weight standards or markers is always included on each gel to enable determination of the size of the proteins.
When all the samples and the markers have been loaded, the apparatus must be connected to the power supply. The lid of the apparatus has built-in electrical connections which are fitted to the appropriate electrode in the gel tank. The cables are then plugged into the power pack, and the current is switched on. The gels are initially run at 100 volts.
Notice the tiny bubbles rising from the electrode. When the current is applied, the samples start to move into the gel. Once the blue dye front has reached the bottom of the stacking gel, the voltage can be increased to 150 volts.
Exact running conditions will vary according to the gel apparatus and the size and thickness of gels. It takes approximately one hour for the dye front to near the bottom of the gel. At this point, the current is switched off, and the apparatus is disassembled. The running buffer is discarded.
At this point, a gel can either be stained to visualise the proteins, or its proteins can be blotted onto a nitrocellulose membrane for detection of a specific protein western blot. Identical samples have been loaded onto the two gels in this experiment so that one gel can be stained and the other can be used for a western blot. We will come back to the western blotting procedure shortly. But first, we will see how it gel is stained.
To recover the gels, the plates are prized apart, leaving the gel stuck to one plate. One small corner of the gel is cut off to indicate the orientation of the gel and to permit identification of the lanes, and the stacking gel is trimmed away. Proteins in the gel are detected by staining with a dye called Coomassie blue dissolved in acidified methanol.
The gel is placed in a container with some of the staining solution and is left to mix gently on a shaking platform for anything from a few hours to overnight. The acidified methanol causes the proteins to precipitate in the gel, and the dye stains them a bright blue. The Coomassie blue stain penetrates the whole gel, but it only binds tightly to the proteins.
Excess dye is washed out by de-staining with an acidified methanol solution. As with the staining step, the gel is agitated gently on the shaker to ensure even de-staining. The result is a gel with protein bands stained blue against a virtually clear background. In this gel, you can clearly see the molecular weight markers and many bands in the sample lanes. Remember that a high molecular weight protein travels a shorter distance down the gel than does a low molecular weight protein.
For purposes of documentation, gels can be either photographed or digitally recorded immediately following de-staining. Alternatively, they can be preserved by drying them between two sheets of clear membrane.
We will now see how the second gel is used for western blotting. The gel must be transferred as soon as possible after electrophoresis to avoid diffusion of the proteins, which would result in blurring. Remember that this is a different gel from the one that was stained. It is not possible to transfer a stained gel.
A sandwich is built up with firstly several pieces of filter paper soaked in a transfer buffer containing tris, glycine, and either ethanol or methanol. The gel with the stacking general removed is placed on the filter paper, noting its orientation. The nitrocellulose filter, which has been pre-wet with transfer buffer, is placed on top of the gel and rolled to ensure a good contact.
To complete the sandwich, a further set of wet filter papers are positioned on top of the filter. The sandwich of filter paper, nitrocellulose, and gel is held together between sponges soaked in transfer buffer and clamped in a cassette. The gel sandwich, clamped in place, is positioned in the transfer tank filled with cold transfer buffer such that the side with the filter faces the anode. Thus, the filter is positioned between the gel and the anode.
Transfer is performed on ice or in a cold room to avoid overheating. The electrodes are connected to the power supply with a constant current of 150 milliamps for overnight transfer or 450 milliamps for transfer in three to four hours.
When transfer is complete, the power supply is switched off and the cassette removed. The sandwich can now be disassembled to recover the nitrocellulose filter. The gel is discarded. To confirm transfer and to identify marker bands, the proteins are temporarily stained on the filter with a dye called Ponceau red. The position of the markers can be permanently identified by marking the filter with a biro.
The filter is now placed in a blocking solution containing phosphate-buffered saline, nonfat milk powder, and Tween 20 detergent to block nonspecific protein binding. The filter is gently agitated on a shaker for one hour.
To minimise the amount of expensive antibodies required, incubation of the filter with the primary antibody is often performed in a sealed plastic bag. The primary antibody specific for the protein of interest is diluted in blocking solution. 5 millilitres of diluted antibody is sufficient for a filter such as this.
The incubation with the primary antibody is for one to two hours at room temperature with mixing. After incubation with the primary antibody, the solution is removed. The filter is returned to a dish for washing. Four or five washes of 10 minutes each in blocking solution is sufficient to wash off unbound primary antibody.
In this experiment, the primary antibody was raised in mouse. The nitrocellulose filter is now incubated with a secondary antibody, which specifically recognises mouse immunoglobulins and which is conjugated to horseradish peroxidase. The filter is incubated with the secondary antibody for one hour with gentle shaking.
After incubation with the secondary antibody, the antibody is removed. The filter is now subjected to four to five 10-minute washes with PBS containing Tween 20.
Detection of the horseradish peroxidase activity associated with the secondary antibody is achieved using a substrate for the enzyme, which is converted into a chemiluminescent product. The filter is incubated for one minute in freshly-prepared substrate. To minimise deterioration of the signal, the filter is protected from excessive exposure to light.
The filter is then drained of substrate and sandwiched between two layers of cling film. The filter is then exposed to photographic film in a film cassette, and the film is developed. Exposure times vary depending on the amount of protein and the avidity of the antibodies.
Dark bands on the photographic film indicate the presence of the protein recognised by the primary antibody. In this case, the positions of the markers have been marked on the photographic film. Compare the stained gel from this experiment with the western blot results. The many proteins evident in the stained gel will also have been transferred to the nitrocellulose from the duplicate gel. Notice, however, that only one band is detected in the western blot.