Notes to accompany the slidecast on theory of SDS PAGE and Western blotting
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1 S317 Biological science: from genes to species Notes to accompany the slidecast on theory of SDS PAGE and Western blotting SDS PAGE SDS PAGE is a standard technique for determining the molecular size of a protein and its polypeptide chain composition. It is also very useful for separating complex mixtures of proteins and gives a good impression of the relative abundance of each protein (Figure 1). Figure 1 Appearance of a polyacrylamide gel stained with Coomassie Blue to detect proteins. The gel has three lanes and the left-hand lane contains molecular weight markers (1 6). The other two lanes contain proteins X and Y. Protein Y runs faster than X and is therefore smaller. The actual values for X and Y would be determined by interpolation from the markers. Molecular mass (M r ) When proteins have been treated with SDS they acquire a uniform negative charge and their mobility in a polyacrylamide gel is dependent on their size small molecules move through the gel more quickly than large molecules. One can estimate the molecular mass of an unknown protein by comparing its mobility with that of molecular weight markers, a collection of proteins of known molecular weight. This provides a value of the relative molecular mass of the protein or M r. At best, an SDS gel can resolve proteins which vary by 2 % in their M r i.e. it is just possible to separate proteins of (e.g.) and Copyright 2015 The Open University Printed in the United Kingdom 1.1
2 Generally, the technique works well for polypeptides of M r , although it is necessary to select an appropriate concentration of polyacrylamide in the gel, according to the size of the proteins under investigation. If, however, the polypeptides have associated carbohydrate (glycoproteins) or lipid groups (lipoproteins), then the M r values derived from SDS PAGE can be significantly different from the true molecular mass of the molecule. You will need to know the method for deriving M r values by interpolation from a standard curve (Figure 2). Figure 2 Derivation of a standard curve for measuring M r by SDS PAGE. To derive a standard curve, the mobility of each marker band is measured relative to the full length of the gel, i.e. the distance travelled by the dye front. In the example above, the known marker protein 3 has travelled 22 mm down the gel, and the dye front is at 82 mm. Therefore, the relative mobility is: R f = 22/82 = The mobilities of all the other markers are derived in the same way and their R f values will lie in the range 0 1. These values are plotted against the log of the molecular weight of each protein, producing a curve which is approximately 2
3 linear. The relative molecular mass (M r ) of an unknown protein X is derived by determining its R f value and then reading its M r from the standard curve. Acrylamide concentration The mobility of proteins in SDS PAGE depends on the concentration of the acrylamide gel. Large proteins will not enter the gel (R f = 0) whereas small proteins will not be retarded at all and will therefore run with the dye front (R f = 1). The range of proteins that are separated in a gel (0 < R f < 1) therefore depends on the gel concentration. As a rule of thumb, the usable separation range of SDS gels is: 5 % gel % gel % gel % gel In practice, one aims to select a gel concentration that places the proteins of interest somewhere in the middle of the range (0.2 < R f < 0.8). In the experiments you will have to select a suitable concentration of acrylamide to separate proteins and you have the option of using a 5%, 8%, 12% or 15% gel. Molecular composition SDS PAGE can provide information on the subunit composition of proteins. Treatment with SDS breaks non-covalent bonds between the amino acid residues, but does not break covalent bonds. Consequently, single proteins may appear as more than one band if the polypeptides are non-covalently linked. Also, if a sample of a protein is prepared unreduced, any disulfide bonds remain intact; conversely, reduction of the protein (e.g. with -mercaptoethanol) causes the disulfide bonds to break and the polypeptides then separate. Comparison of the pattern of bands on a reduced and unreduced gel can therefore be very informative. For example, a band for a single protein of M r is present on an unreduced gel. If this band disappears on a reduced gel, to be replaced by bands of M r = 8000, and , it indicates that the M r protein consists of three disulfide-linked polypeptides. Notice how the M r values of the components add up to the M r value of the protein. Consequently, it is also possible to infer something about the stoichiometry of a protein. For example, if a protein of M r = produces a band of M r = on a reduced gel, it suggests that it consists of two disulfide-linked polypeptides. In the experiments here, the samples are treated with a buffer containing -mercaptoethanol, so that the disulfide bonds are reduced and individual polypeptide chains are separated. Molecular abundance and detection limit When proteins have been separated in an SDS gel, they are most often visualised by staining with Coomassie Blue. This binds to all proteins, roughly in proportion to their abundance, which means that a strongly-stained band on the gel contains more protein than a weakly-stained band. Coomassie Blue can detect bands which contain less than 1 g protein. Although it depends on the type of gel and the proteins, the range of detection is typically 0.2 g (weak band) to 50 g (very strong band) protein. The detection limit for Coomassie stain is therefore said to be 0.2 g per band. Sample preparation Sample preparation for SDS PAGE (and Western blotting) involves heating the proteins to 95 C in a sample buffer containing SDS and a dye, bromophenol blue, that runs at the solvent front, indicating the total length run by the samples (R f = 1). 3
4 The sample buffer also contains glycerol or sucrose to increase the sample density, allowing the sample to sink into the wells. The sample buffer may also contain -mercaptoethanol to reduce the proteins as described above. Before adding the sample buffer, it is necessary to dilute samples to a known protein concentration. Firstly, this ensures that there is enough protein to detect and that the gel is not overloaded. Secondly, where samples are to be compared, it is essential that the total protein concentration in each sample/well is identical. Since samples derived from different sources will have different concentrations, they must be diluted to an equal concentration before adding sample buffer. Western blotting Although SDS PAGE tells us a lot about the size and composition of a protein, it cannot tell us anything about its identity or function. Western blotting (also called immunoblotting) is used to identify individual molecules or polypeptides that have been resolved by SDS PAGE. The technique uses antibodies to identify individual proteins, polypeptides or even particular molecular structures (e.g. phosphorylated tyrosine residues) on filters which have been blotted from SDS gels (Figure 3). Figure 3 Western blotting. Following SDS PAGE, polypeptides are transferred (blotted) onto a nitrocellulose filter by applying an electrical current across the gel. Unoccupied sites on the filter are blocked by incubation with non-specific proteins before incubation with a specific primary antibody that recognises the protein of interest. Unbound primary antibody is washed off and the filter incubated with an enzyme-linked secondary antibody that binds to the primary antibody. The secondary antibody and the enzyme is detected either by i) a substrate which deposits a coloured end product onto the filter, or ii) a substrate that generates light (chemiluminescence) in the presence of the enzyme this can be detected photographically or with suitable imaging instruments. Other alternatives are the use of fluorescent or radioactive secondary antibodies. Selection of primary antibodies The technique is described in some detail in the theory section of the slidecast. One fact that you should note, however, is that antibodies recognise part of the three-dimensional structure of their target molecule, and the preparation of proteins for SDS PAGE denatures them. Sometimes, therefore, an antibody which binds to a native protein will not be able to recognise a protein which has been run on an SDS gel and blotted; other times, there is sufficient structure remaining for the antibody to recognise the protein and so it can be used for Western blotting often it is just a case of trying it out to see if it works. 4
5 In some cases, an antibody will recognise one polypeptide chain from a protein, but not another. Even antibodies that recognise the protein on a Western blot may vary in their suitability; antibodies may not be totally specific for their target protein. For example, if an antibody recognises a three-dimensional structure present on an off-target protein, it is said to be cross-reactive. In this case, the primary antibody will produce bands both with the target protein and with one or more other off-target proteins. Such activity is called non-specific binding. For these reasons, primary antibodies are selected not just for their ability to recognise the target protein in a Western blot, but also for their specificity i.e. they do not recognise other proteins in the samples. In the first experiment, you will be asked to identify a suitable primary antibody that will be used in later experiments. Concentration of primary antibodies and detection limits The concentration of primary antibody used in the Western blot is another important consideration. If the concentration is too low, there will not be enough to detect the target protein on the blot. If the antibody concentration is too high, the background staining of the blank areas of the blot increases and the differential between the specific bands and background is reduced. Moreover, expensive antibody is wasted. For these reasons, it is essential to find a suitable concentration of the primary antibody. Concentrations between 250 ng/ml and 5 g/ml are usually suitable. In the second experiment, you will have to find a suitable concentration of antibody to detect the target protein. In Western blotting, the detection limit for a protein on a blot depends on the detection method used. Systems based on fluorescent or radioactive secondary antibodies are very sensitive. The virtual laboratory simulates detection by chemiluminescence, and the sensitivity of this technique can be adjusted according to the time allowed to detect the emitted light (i.e. a long exposure will be more sensitive than a short exposure). 5
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