As mentioned previously, polyacrylamide gels can be used for the separation and analysis of proteins and relatively small nucleic acid molecules. For example, when it was first invented, Sanger’s DNA sequencing method (see in details in Chapter 10) applied PAGE to separate linear single-stranded DNA molecules based on their length. The resolution of the PAGE method is so high that, in the size range of about 10-1000 nucleotide units, it is capable of separating DNA molecules that differ in length only by a single monomer unit. In the case of single-stranded DNA, individual molecules are separated solely based on their length. This is due to the fact that, in the case of DNA (or RNA), the number of negative charges is a simple linear function of the number of monomer units (i.e. the length of the molecule). In other words, the specific charge (number of charges per particle mass) is invariant, i.e. it is the same for all DNA molecules. It is so because each monomer unit has one phosphate moiety that carries the negative charge. When an appropriate denaturing agent, such as urea, is added to the DNA sample and the gel is heated, the shape of the varying-length linear DNA molecules becomes identical. As a consequence, denatured molecules will be separated exclusively based on their size. (We will see the same principle at the SDS-PAGE method that separates denatured proteins almost exclusively based on their size (molecular weight)). There are several PAGE methods (SDS-PAGE, isoelectric focusing, 2D PAGE) that can be applied mostly for the separation of proteins based on distinct molecular properties.
At a given pH, different proteins carry different amounts of electric charge. Moreover, different proteins have different shapes and sizes, too. Consequently, during electrophoresis, proteins are separated by a complex combination of their charge, shape and size. PAGE separation of proteins provides high resolution. However, as three independent molecular properties simultaneously influence electrophoretic mobility, it will provide limited room for precise interpretation. For example, when two proteins are compared, it remains hidden what makes one of them migrate faster: a larger number of electric charges, a smaller size, or a more spherical shape. Nevertheless, even the simplest PAGE method, which will be referred to as native PAGE, provides many particular advantages (see below).
In order to increase the analytical applicability of the PAGE technology, several variations of the method have been established to separate proteins based on a single molecular property. As we will see, SDS-PAGE separates proteins based primarily on molecular weight, while isoelectric focusing separates proteins exclusively based on isoelectric point.
In the presence of suitable initiator and catalyst compounds, acrylamide can readily polymerise in a radical process. (Acrylamide is harmful by inhalation or skin contact, and thus it should be handled with care.) This reaction would lead to very long polyacrylamide chains, yielding a highly viscous liquid instead of a gel. As already mentioned, these long chains need to be cross-linked to form a three-dimensional network. This is achieved by mixing N,N'-methylenebisacrylamide into the acrylamide solution. In essence, N,N'-methylenebisacrylamide is composed of two acrylamide molecules covalently interconnected via a methylene moiety. When, during the polymerisation reaction, the acrylamide groups of N,N'-methylenebisacrylamide molecules become incorporated in the long polyacrylamide chains, cross-links are formed between the polyacrylamide chains leading to a gel (Figure 7.2). In the course of electrophoresis, ions (proteins or nucleic acids) are separated in this gel.
Without any modification, polyacrylamide electrophoresis separates macromolecular ions based on a combination of charge, size and shape. Size (and shape) separation is due to the molecular sieving property of the gel. The size range in which molecules can be separated is dictated by the average pore size of the gel. In the case of polyacrylamide gels, this can be controlled through the concentration of the acrylamide monomer and the proportion of the cross-linking N,N'-methylenebisacrylamide. The acrylamide concentration can be set in the range of about 4-20 % as this is the range in which the mechanical properties of the gel are appropriate. Below this range the gel will be too soft and it will not keep its shape, while above this range it will be too rigid and prone to break. The optimal proportion of the N,N'-methylenebisacrylamide component is 1-3 % relative to the acrylamide component. The polyacrylamide gel possesses all advantageous properties necessary for a good electrophoresis medium, i.e. it is hydrophilic, free of electric charges and chemically stable. A further very important property of the polyacrylamide gel is that it does not participate in any non-specific or specific binding interaction with proteins. Furthermore, the polyacrylamide gel does not interfere with common protein staining reactions.
When electrophoresis is performed under native (non-denaturing) conditions, such as near neutral pH and ambient or lower temperature, many enzymes retain their native conformation and, in turn, their enzymatic activity. This way, many enzymes can be separated and specifically detected in the gel after electrophoretic separation.
In the course of creating the gel, a buffer with a properly chosen pH is mixed into the acrylamide/N,N'-methylenebisacrylamide solution. Radical polymerisation is subsequently triggered by suitable catalyst and initiator compounds. The catalyst is usually ammonium persulfate, which spontaneously decomposes in aqueous media, thereby generating free radicals. These free radicals in themselves cannot efficiently cleave the double bonds of the acrylamide molecule, but are able to excite the electrons of the initiator molecules. This leads to the generation of free radicals, originating from the initiator molecules, that are able to trigger radical polymerisation of acrylamide monomers. The most frequently used initiator is tetramethylethylenediamine (TEMED).
There are two types of gels according to their geometry. In early gel electrophoretic applications, gel tubes were used that allowed only a single sample to be run. Gel slabs were later introduced, allowing for many samples to be run at the same time in the same gel in parallel. Gel slabs became much more common than gel tubes. Gel slabs are created by pouring the gel-forming solution between two parallel glass sheets prior to polymerisation (Figure 7.3). Besides its higher throughput, this gel geometry provides another important advantage over gel tubes: samples are loaded side by side on such slabs and are run in the same gel at the same time. This allows for a more reliable comparison of the samples, facilitating the interpretation of experimental results.
Proper selection of pH and acrylamide concentration is instrumental for successful electrophoresis. For protein electrophoresis, the pH is set usually higher than the pI value of the proteins in the sample. At such a pH, all proteins will be negatively charged and will move towards the anode. The buffer in the medium serves two purposes. One is to set and maintain the proper pH during electrophoresis. The other function of the buffer is to establish the electric current in the medium.
The majority of the electric current is carried by the ions of the buffer. Normally, the protein-ions that are separated by electrophoresis have only a negligible contribution to the current. In other words, proteins have a low ion transport number. However, if the buffer concentration is set too low, the contribution of proteins to carrying the current will increase, and the protein molecules will migrate rapidly. This usually leads to smearing of the bands of migrating proteins. On the other hand, if the buffer concentration is set too high, the mobility of the proteins will be too low. In this case the electrophoresis process would take a very long time. Unnecessary lengthening of the process provides excess time for diffusion, which lowers the resolution of separation.
According to the applied buffer system, gel electrophoretic methods can be classified into two types: continuous and discontinuous. Continuous methods apply the same buffer in the gel and in the two buffer chambers containing the electrodes. The only advantage of this method lies in its simplicity. More complex discontinuous methods were introduced to provide higher resolution. SDS polyacrylamide gel electrophoresis (see later) is usually associated with such a discontinuous system.
The discontinuous system applies two gels of different pore size and three different buffers. One of the gels, the resolving gel, is polymerised at a higher acrylamide concentration. The pore size of this gel is set according to the size range of the proteins to be separated. Another gel, the stacking gel is created on top of the resolving gel. (The gels are mounted in a vertical format.) The stacking gel is polymerised from a more dilute acrylamide solution to provide larger pores. This pore size does not provide a molecular sieving effect.
As mentioned above, there are three buffers: different ones in each of the two gels and a third one, the so-called ‘running buffer’ in the buffer chambers containing the electrodes. In the gel buffers, the anion originates from a strong acid; it is usually chloride ion. Dissociation of strong acids does not depend on the pH: these acids always fully dissociate. Consequently, chloride ion is never protonated in the solution: its ionisation state is independent of the pH. On the other hand, the anion component of the running buffer is the conjugate base of a weak acid. Consequently, the ionisation state of this ion depends on the pH of the buffer. Glycinate ion is one of the most frequently used compounds for this purpose. The pH in the running buffer is set to 8.3.
The protein sample is layered on the top of the stacking gel. When an electric field is generated by the power supply, the protein ions and the ions of the running buffer enter the stacking gel. The pH in the stacking gel is set to 6.8. This value is only slightly higher than the pI value of glycine (6.5). At this pH, most glycine molecules are in a neutral zwitterionic state, and only a small portion of the molecules carry a net negative charge. In this state, glycine has a low electrophoretic mobility and a corresponding low transport number. The local sparsity of ions elevates the local electric resistance of the medium. As the electric current must be of the same magnitude at any segments of the electric circuit (there is no macroscopic charge separation), the voltage will increase according to Ohm’s law. Due to this effect, the migration speed of the proteins will be relatively high and the protein front will reach the chloride front in the stacking gel. The ion concentration in the chloride front is high and, therefore, here the electric resistance and the voltage are low. This slows down the protein front. This effect results in a very sharp protein front, with the protein molecules being crowded right behind the chloride ion front.
The protein sample will thus enter the resolving gel in a sharp band. The pH in the resolving gel is set to about 8.8. At this pH, almost all glycinate molecules are in the anionic state. Thus, the electric mobility of glycinate increases, and the concentrating effect applied by the stacking gel ends in the resolving gel. Different proteins will be separated in the resolving gel according to their charge, size and shape.
In most electrophoretic methods, a tracking dye is mixed in the sample. Usually, this dye is chosen to have a higher electrophoretic mobility than any of the components of interest (proteins or nucleic acids) in the sample. The function of the tracking dye is to visualise the running front and, in turn, the completeness of the run. The most popular tracking dye is bromophenol blue.
The following sections review the various PAGE methods listed from the simplest to the most complex one.
Native PAGE is an electrophoresis method to separate native proteins. The conditions are set such that the migrating proteins are kept in their native state. The buffers provide a non-denaturing, native-like milieu, and the electrophoresis is performed at low temperature in order to dissipate heat. Many enzymes retain their native conformation and their enzymatic activities while running in the gel. If certain conditions apply, these enzymes can be highly selectively detected within the gel through a specific ‘staining’ reaction even in the presence of a large excess of ‘contaminating’ proteins. After completion of electrophoresis, the gel is soaked in a solution containing the substrate of the enzyme. As the substrate is usually a small molecule, it quickly diffuses into the gel while the large enzyme molecules do not diffuse out. In an optimal case, the natural product of the enzymatic reaction is a coloured and insoluble compound that precipitates inside the gel and marks the exact location of the enzyme. Of course, most enzymes do not have such natural substrates. However, once the molecular mechanism of catalysis is revealed, synthetic substrates can be designed that, on the one hand, mimic natural substrates and, on the other hand, lead to colourful insoluble products.
Native PAGE is also a useful method for checking the uniformity of the isolated protein. Even if the purified protein sample contains only a single type of protein, the sample might not be uniform. Some of the molecules might be unfolded or have undergone chemical modifications. Unfolding changes the overall shape of the molecule, while most chemical modifications change the electric charge of native molecules. These alterations can be detected after traditional staining of the purified sample. If no such side products are present, protein molecules will run in a single sharp band. Otherwise, multiple bands or smearing of the band is expected.
In addition, native PAGE can also be used to detect complex formation between proteins. If two (or more) proteins (or proteins and non-proteinous ligands) form a complex, the complex can be detected as an extra band in the gel. This is because in native-like conditions, many non-covalent (subunit-subunit, receptor-ligand, enzyme-inhibitor) interactions are maintained and the complex migrates apparently as a single molecule.
In the course of native PAGE, it is highly important to pay attention to the relationship of the pI values of the proteins or protein complexes and the pH of the gel buffer, as this will determine where individual proteins will migrate in the gel.
SDS-PAGE is an electrophoresis method to separate proteins. However, unlike in the case of native PAGE, here the proteins migrate in their denatured state. As it was mentioned in the general introduction to traditional (native) PAGE, the migration velocity of proteins is a function of their size, shape and the number of electric charges they carry. As the velocity is a complex function of these properties, native PAGE cannot be used to estimate the molecular mass of proteins. The traditional native PAGE method is similarly unable to assess whether a purified protein is composed of a single subunit or multiple subunits. Even a multi-subunit protein may migrate in a single sharp band.
SDS-PAGE (Figure 7.4) was introduced to analyse such cases and to allow the estimation of the molecular mass of single-subunit proteins or those of individual subunits of multi-subunit proteins. SDS-PAGE is the most prevalent PAGE method currently in use.
SDS (sodium dodecyl sulphate) is an anionic detergent. When proteins are treated with SDS at high temperature, radical conformational changes occur. The treatment breaks all native non-covalent intermolecular (inter-subunit) and intramolecular interactions. The subunit structure of multi-subunit proteins disintegrates and the proteins unfold. If the native structure is stabilised by disulfide bridges, reducing agents are also added to open up these connections. SDS molecules bind to unfolded proteins in large excess, providing extra negative charges to the molecules.
The amount of the bound SDS molecules is largely independent of the amino acid sequence of the polypeptide chain and it is roughly a linear function of polypeptide length—i.e. the molecular mass of the protein. Therefore, upon SDS-treatment, the specific charge (the charge-to-mass ratio) of different proteins will become roughly identical. Another result of the treatment is that the shape of the different proteins becomes similar. The negatively charged SDS molecules repel each other, which lends a (presumably) rod-like shape to the SDS-treated proteins. These factors together result in a situation analogous to the one already discussed in this chapter for the PAGE separation of linear single-stranded (denatured) DNA molecules. Instead of being separated simultaneously by charge, shape and size, SDS-treated proteins—just like denatured linear DNA molecules—will be separated solely based on their size. As size is a linear function of mass, SDS-PAGE ultimately separates proteins based on their molecular mass.
SDS-PAGE is the most popular cost-effective method to estimate the molecular mass of protein subunits with considerable accuracy. The relative mobility (i.e. the running distance of the protein divided by the running distance of the tracking dye) of the SDS-treated protein is in inverse linear proportion to the logarithm of the molecular mass of the protein. By running several proteins of known molecular mass simultaneously alongside the protein of interest, a log molecular mass – relative mobility calibration curve (a descending linear graph) can be created. Based on the calibration curve, the estimated molecular mass of the protein in question can be easily calculated.
Table 7.I below shows the useful separating range of polyacrylamide gels as a function of acrylamide concentration. In the useful range, the log molecular mass – relative mobility relationship is linear.
Linear range of separation
Table 7.I. Relation between acrylamide concentration and the molecular mass of optimally separated molecules
SDS-PAGE is a standard method for assessing whether the sample of an isolated protein is homogeneous. Besides that, SDS-PAGE is a robust method for the analysis of large supramolecular complexes such as multi-enzyme complexes or the myofibril, as discussed below. SDS-PAGE separates and denatures individual subunits of these complexes. Thus, all polypeptide chains will migrate separately in the gel. Via various staining procedures, all subunits can be visualised and the relative amounts of these proteins (subunits) can also be determined. This allows for the identification of each subunit of a complex and provides a good estimate of the stoichiometry of subunits, too.
In the course of isoelectric focusing, the conditions are set in a way that proteins will be separated exclusively based on their isoelectric point (Figure 7.5). The two termini and many side chains of proteins contain dissociable groups (weak acids or bases). The dissociation state of these groups is a function of the pH of the environment (as described quantitatively by the Henderson-Hasselbalch equation, see Chapter 3). Isoelectric focusing is based on the pH-dependent dissociation of these groups. Due to this pH-dependent phenomenon, the net electric charge of a protein molecule will be a function of the pH of the medium. If, in a given protein, the number of acidic residues (Asp, Glu) exceeds that of the basic ones (Arg, Lys, His), the protein will have a net negative charge at neutral pH. The isoelectric point (pI) of the protein—i.e. the pH at which the net charge of the protein is zero—will be in the acidic pH range. Such proteins are often denoted as acidic proteins. If the number of basic residues exceeds that of the acidic ones, the protein will be positively charged at neutral pH, and its pI value will be in the basic pH range. These proteins are often called basic proteins.
Isoelectric focusing is an efficient high-resolution method because the pI values of various proteins are spread across a broad range. If the pH is lower than the pI of the protein, the protein will be positively charged and will move towards the cathode during electrophoresis. If the pH is higher than the pH of the protein, the protein will be negatively charged and will migrate towards the anode. If the pH equals the pI value, the net charge of the protein will be zero and the protein will not migrate in the gel any further.
In the course of isoelectric focusing, proteins are placed in a gel representing a special medium in which the pH gradually decreases by going from the negative cathode towards the positive anode. As the protein migrates, it encounters a gradually changing pH and its net charge will also change accordingly. If it has a net negative charge and therefore moves towards the cathode, it will encounter a gradually decreasing pH, i.e. a more and more acidic environment. Consequently, the protein will take on more and more protons—up to a level where its net charge will be zero. This state is reached when the protein reaches a location where the pH equals its pI value. At this point, the protein will stop moving because no electric force will be exerted on it. If it spontaneously diffused further towards the anode, it would take on more protons, would become positively charged and would turn back to migrate towards the cathode. Following the same line of thinking, if a positively charged protein moves towards the cathode, it will encounter increasing pH and lose more and more protons. It will migrate to the place where the pH equals its pI value and will thus stop. If it diffused further towards the cathode, it would become negatively charged and would turn back towards the anode. As one can see, by performing electrophoresis in a medium in which the pH decreases from the cathode towards the anode, each protein will “find its place” according to its pI value and will become sharply focused at that location. In addition, it does not matter where exactly the proteins were introduced in the medium between the cathode and the anode.
A decisive component of this method is the usually linear pH gradient created inside the gel. There are two methods to create such a gradient. One of them applies carrier ampholytes (ampholyte is an acronym from the words amphoteric and electrolyte). Ampholytes or zwitterions are molecules that contain both weakly acidic and weakly basic groups. Just like in the case of proteins, the net charge of ampholytes is a function of the pH. In the course of isoelectric focusing, a mixture of various ampholytes is used such that the pI of the various ampholyte components will cover a range in which the pI values of the “neighbouring” ampholytes differ only slightly. This ampholyte mixture is soaked in the gel and an appropriate electric field is generated by a power supply. This leads to a process analogous to the one already explained for proteins. Each ampholyte will migrate to the location where its net charge becomes zero. As soon as this steady-state is achieved, ampholytes will function as buffers and keep the pH of their immediate environment constant. This establishes the pH gradient in which the proteins can be separated.
The other, more sophisticated method applies special ampholytes that can be covalently polymerised into the polyacrylamide gel. The appropriate ampholyte gradient is created before the gel is polymerised. This way, the gradient will be covalently fixed in the gel, providing an immobilised pH gradient. The appropriate pH range provided by the ampholyte mixture should be selected based on the pI values of the proteins to be separated.
Regardless of how the pH gradient was created, once the proteins reach the location in the gel where the pH equals their pI, they finally stop moving and the system reaches a steady-state.
One of the potential technical difficulties encountered during isoelectric focusing originates from the fact that the solubility of proteins is lowest at their pI value (see Chapter 5). This can lead to the precipitation of some proteins in the gel. To prevent this unwanted process, urea is most often applied in the gel as an additive. Urea denatures proteins and keeps denatured proteins in solution. As the pI value of proteins is largely independent of their conformational state, this modification does not compromise the method. The solubility of membrane proteins can be further promoted by the addition of non-ionic detergents.
Isoelectric focusing is aimed at separating proteins based exclusively on their pI value—thus, independently of their size. Therefore, the molecular sieving property of the gel in this method should be avoided. The only function of the gel is to prevent free convectional flows in the medium. Accordingly, for isoelectric focusing, polyacrylamide gels are made at very low acrylamide concentrations, and sometimes even agarose gels are applied when very large pores are needed. Isoelectric focusing is usually performed in a horizontally-mounted electrophoresis apparatus and by applying intense cooling.
The various separation methods are all aimed at separating complex systems to individual components. Separation is always based on at least one physicochemical property that shows diversity among the components. The general problem encountered in the case of complex mixtures is that not all components differ significantly from all other components when only one property is considered. Accordingly, separation based on a single property rarely results in single-component fractions. Some components will be efficiently separated from all others, while some other components will remain in the mixture.
The remaining mixtures can be further fractionated by another separation technique that relies on a different physicochemical property. The most effective separation can be achieved if the combined consecutive separation steps rely on absolutely independent physicochemical properties. A good example of this is the very high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) that combines two already discussed electrophoresis methods, isoelectric focusing and SDS-PAGE (Figure 7.6).
As the first step of 2D gel electrophoresis, isoelectric focusing is performed to separate proteins based on their pI values. Only a single sample is loaded on a gel strip in this step. The sample is separated in one dimension both in a primary and in a figurative sense. In a primary sense because the components are separated along a single line, and in a figurative sense as the separation is based on a single well-defined property, the pI value.
After the first separation step has been completed in the first dimension, the gel strip is soaked in an SDS solution and is fitted tightly to one side of a “classical” SDS polyacrylamide gel. The second separation step is traditional SDS-PAGE, which separates proteins based on their molecular mass. This second step represents a second dimension in both a primary and a figurative sense. The second separation is performed in a second dimension in a direction rectangular to that of the first separation, and the property utilised in the second step (molecular mass) is completely independent of the one utilised in the first step (pI).
If, after the first step, some gel regions contain different proteins that coincidentally have identical pI values, these proteins will be separated from each other in the second step if their molecular mass is different. Note that every aspect discussed for SDS-PAGE also applies to the second separation step of 2D-PAGE. Van der Waals interactions that might have held protein subunits together in the course of isoelectric focusing will break and individual subunits will become separated. If disulfide bridges need to be opened up, some kind of reducing agent needs to be added. Accordingly, in the second separation step, single polypeptide chains will migrate in the gel. If isoelectric focusing collects a multimeric protein at a certain gel location, the second electrophoresis step will dissect it into individual chains. If the multimer contains subunits of different sizes, these subunits will be separated from each other in the second separation step.