# Electrophoresis of proteins
## SDS-PAGE

SDS-PAGE is a form of polyacrylamide gel electrophoresis (PAGE) which is commonly used to separate proteins according to their molecular weight. Sodium dodecyl sulfate (SDS) is an anionic detergent that denatures secondary and non–disulfide–linked tertiary structures, and additionally applies a negative charge to each protein in proportion to its mass. In addition to SDS, proteins may optionally be briefly heated to near boiling in the presence of a reducing agent, such as dithiothreitol (DTT) or 2-mercaptoethanol (beta-mercaptoethanol/BME), which further denatures the proteins by reducing disulfide linkages, thus overcoming some forms of tertiary protein folding, and breaking up quaternary protein structure (oligomeric subunits). This is known as reducing SDS-PAGE.

### Polyacrylamide gels

The gels typically consist of acrylamide, bisacrylamide, the denaturant (SDS), and a buffer with an adjusted pH. The solution may be degassed under a vacuum to prevent the formation of air bubbles during polymerization. Alternatively, butanol may be added to the resolving gel (for proteins) after it is poured, as butanol removes bubbles and makes the surface smooth. A source of free radicals and a stabilizer, such as ammonium persulfate and TEMED are added to initiate polymerization. The polymerization reaction creates a gel because of the added bisacrylamide, which can form cross-links between two polyacrylamide molecules. The ratio of bisacrylamide to acrylamide can be varied for special purposes, but is generally about 1 part in 35. The acrylamide concentration of the gel can also be varied, generally in the range from 5% to 25%. Lower percentage gels are better for resolving very high molecular weight molecules, while much higher percentages are needed to resolve smaller proteins.
Gels are usually polymerized between two glass plates in a gel caster, with a comb inserted at the top to create the sample wells. After the gel is polymerized the comb can be removed and the gel is ready for electrophoresis.

### Electrophoresis

Various buffer systems are used in PAGE depending on the nature of the sample and the experimental objective. The buffers used at the anode and cathode may be the same or different.
An electric field is applied across the gel, causing the negatively charged proteins or nucleic acids to migrate across the gel from the negative electrode (the cathode) towards the positive electrode (the anode). Depending on their size, each biomolecule moves differently through the gel matrix: small molecules more easily fit through the pores in the gel, while larger ones have more difficulty. The gel is run usually for a few hours, though this depends on the voltage applied across the gel; migration occurs more quickly at higher voltages, but these results are typically less accurate than at those at lower voltages. After the set amount of time, the biomolecules have migrated different distances based on their size. Smaller biomolecules travel farther down the gel, while larger ones remain closer to the point of origin. Biomolecules may therefore be separated roughly according to size, which depends mainly on molecular weight under denaturing conditions, but also depends on higher-order conformation under native conditions. However, certain glycoproteins behave anomalously on SDS gels.
Similarly, as in nucleic acid gel electrophoresis, tracking dye is often used. Anionic dyes of a known electrophoretic mobility are usually included in the sample buffer. A very common tracking dye is Bromophenol blue. This dye is coloured at alkali and neutral pH and is a small negatively charged molecule that moves towards the anode. Being a highly mobile molecule it moves ahead of most proteins.

### Discontinuous SDS-PAGE

Most protein separations are performed using a "discontinuous" (or DISC) buffer system that significantly enhances the sharpness of the bands within the gel. During electrophoresis in a discontinuous gel system, an ion gradient is formed in the early stage of electrophoresis that causes all of the proteins to focus into a single sharp band. The formation of the ion gradient is achieved by choosing a pH value at which the ions of the buffer are only moderately charged compared to the SDS-coated proteins. These conditions provide an environment in which Kohlrausch's reactions determine the molar conductivity. As a result, SDS-coated proteins are concentrated to several fold in a thin zone of the order of 19 μm within a few minutes. At this stage all proteins migrate at the same migration speed by isotachophoresis. This occurs in a region of the gel that has larger pores so that the gel matrix does not retard the migration during the focusing or "stacking" event. Separation of the proteins by size is achieved in the lower, "resolving" region of the gel. The resolving gel typically has a much smaller pore size, which leads to a sieving effect that now determines the electrophoretic mobility of the proteins. At the same time, the separating part of the gel also has a pH value in which the buffer ions on average carry a greater charge, causing them to "outrun" the SDS-covered proteins and eliminate the ion gradient and thereby the stacking effect.
A very widespread discontinuous buffer system is the tris-glycine or "Laemmli" system that stacks at a pH of 6.8 and resolves at a pH of ~8.3-9.0. A drawback of this system is that these pH values may promote disulfide bond formation between cysteine residues in the proteins because the pKa of cysteine ranges from 8-9 and because reducing agent present in the loading buffer doesn't co-migrate with the proteins. Recent advances in buffering technology alleviate this problem by resolving the proteins at a pH well below the pKa of cysteine (e.g., bis-tris, pH 6.5) and include reducing agents (e.g. sodium bisulfite) that move into the gel ahead of the proteins to maintain a reducing environment. An additional benefit of using buffers with lower pH values is that the acrylamide gel is more stable at lower pH values, so the gels can be stored for long periods of time before use.
As voltage is applied, the anions (and negatively charged sample molecules) migrate toward the positive electrode (anode) in the lower chamber, the leading ion is Cl− (high mobility and high concentration); glycinate is the trailing ion (low mobility and low concentration). SDS-protein particles do not migrate freely at the border between the Cl− of the gel buffer and the Gly− of the cathode buffer. Friedrich Kohlrausch found that Ohm's law also applies to dissolved electrolytes. Because of the voltage drop between the Cl− and Glycine-buffers, proteins are compressed (stacked) into micrometer thin layers. The boundary moves through a pore gradient and the protein stack gradually disperses due to a frictional resistance increase of the gel matrix. Stacking and unstacking occurs continuously in the gradient gel, for every protein at a different position. For a complete protein unstacking the polyacrylamide-gel concentration must exceed 16% T. The two-gel system of "Laemmli" is a simple gradient gel. The pH discontinuity of the buffers is of no significance for the separation quality, and a "stacking-gel" with a different pH is not needed.

### Staining

Coomassie Brilliant Blue (CBB) is the most popular protein stain. It is an anionic dye, which non-specifically binds to proteins. The structure of CBB is predominantly non-polar, and it is usually used in methanolic solution acidified with acetic acid. Proteins in the gel are fixed by acetic acid and simultaneously stained. The excess dye incorporated into the gel can be removed by destaining with the same solution without the dye. The proteins are detected as blue bands on a clear background. As SDS is also anionic, it may interfere with staining process. Therefore, large volume of staining solution is recommended, at least ten times the volume of the gel.
Coomassie Brilliant Blue G-250 differs from Coomassie Brilliant Blue R-250 by the addition of two methyl groups. The suffix "R" in the name of Coomassie Brilliant Blue R-250 is an abbreviation for Red as the blue colour of the dye has a slight reddish tint. For the "G" variant the blue colour has a more greenish tint. The "250" originally denoted the purity of the dye.
Silver staining is used when more sensitive method for detection is needed, as classical Coomassie Brilliant Blue staining can usually detect a 50 ng protein band, Silver staining increases the sensitivity typically 50 times. The exact chemical mechanism by which this happens is still largely unknown. Silver staining was introduced by Kerenyi and Gallyas as a sensitive procedure to detect trace amounts of proteins in gels. The technique has been extended to the study of other biological macromolecules that have been separated in a variety of supports. Many variables can influence the colour intensity and every protein has its own staining characteristics; clean glassware, pure reagents and water of highest purity are the key points to successful staining. Silver staining was developed in the 14th century for colouring the surface of glass. It has been used extensively for this purpose since the 16th century. The colour produced by the early silver stains ranged between light yellow and an orange-red. Camillo Golgi perfected the silver staining for the study of the nervous system. Golgi's method stains a limited number of cells at random in their entirety.

## Isoelectric focusing

Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI). It is a type of zone electrophoresis, usually performed on proteins in a gel, that takes advantage of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings.
IEF involves adding an ampholyte solution into immobilized pH gradient (IPG) gels. IPGs are the acrylamide gel matrix co-polymerized with the pH gradient, which result in completely stable gradients except the most alkaline (>12) pH values. The immobilized pH gradient is obtained by the continuous change in the ratio of Immobilines. An Immobiline is a weak acid or base defined by its pK value.
A protein that is in a pH region below its isoelectric point (pI) will be positively charged and so will migrate towards the cathode (negatively charged electrode). As it migrates through a gradient of increasing pH, however, the protein's overall charge will decrease until the protein reaches the pH region that corresponds to its pI. At this point it has no net charge and so migration ceases (as there is no electrical attraction towards either electrode). As a result, the proteins become focused into sharp stationary bands with each protein positioned at a point in the pH gradient corresponding to its pI. The technique is capable of extremely high resolution with proteins differing by a single charge being fractionated into separate bands.
Molecules to be focused are distributed over a medium that has a pH gradient (usually created by aliphatic ampholytes). An electric current is passed through the medium, creating a "positive" anode and "negative" cathode end. Negatively charged molecules migrate through the pH gradient in the medium toward the "positive" end while positively charged molecules move toward the "negative" end. As a particle moves towards the pole opposite of its charge it moves through the changing pH gradient until it reaches a point in which the pH of that molecules isoelectric point is reached. At this point the molecule no longer has a net electric charge (due to the protonation or deprotonation of the associated functional groups) and as such will not proceed any further within the gel. The gradient is established before adding the particles of interest by first subjecting a solution of small molecules such as polyampholytes with varying pI values to electrophoresis.
The method is applied particularly often in the study of proteins, which separate based on their relative content of acidic and basic residues, whose value is represented by the pI. Proteins are introduced into an Immobilized pH gradient gel composed of polyacrylamide, starch, or agarose where a pH gradient has been established. Gels with large pores are usually used in this process to eliminate any "sieving" effects, or artifacts in the pI caused by differing migration rates for proteins of differing sizes. Isoelectric focusing can resolve proteins that differ in pI value by as little as 0.01.

## Two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to separate mixtures of proteins by two properties in two dimensions.
2-D electrophoresis begins with 1-D electrophoresis but then separates the molecules by a second property in a direction 90 degrees from the first. In 1-D electrophoresis, proteins (or other molecules) are separated in one dimension, so that all the proteins/molecules will lie along a lane but that the molecules are spread out across a 2-D gel. Because it is unlikely that two molecules will be similar in two distinct properties, molecules are more effectively separated in 2-D electrophoresis than in 1-D electrophoresis.
2-D electrophoresis typically starts with isoelectric focusing. Afterwards, the proteins are treated with sodium dodecyl sulfate (SDS) and separated by their mass at a 90° angle from the first field.

## Native gels

Native gels are run in non-denaturing conditions, so that the analyte's natural structure is maintained. This allows the physical size of the folded or assembled complex to affect the mobility, allowing for analysis of all four levels of the biomolecular structure. For biological samples, detergents are used only to the extent that they are necessary to lyse lipid membranes in the cell. Complexes remain—for the most part—associated and folded as they would be in the cell. One downside, however, is that complexes may not separate cleanly or predictably, as it is difficult to predict how the molecule's shape and size will affect its mobility.
Unlike denaturing methods, native gel electrophoresis does not use a charged denaturing agent. The molecules being separated (usually proteins or nucleic acids) therefore differ not only in molecular mass and intrinsic charge, but also the cross-sectional area, and thus experience different electrophoretic forces dependent on the shape of the overall structure. For proteins, since they remain in the native state they may be visualised not only by general protein staining reagents but also by specific enzyme-linked staining.

### Blue native PAGE

BN-PAGE is a native PAGE technique, where the Coomassie Brilliant Blue dye provides the necessary charges to the protein complexes for the electrophoretic separation. The disadvantage of Coomassie is that in binding to proteins it can act like a detergent causing complexes to dissociate. Another drawback is the potential quenching of chemoluminescence (e.g. in subsequent western blot detection or activity assays) or fluorescence of proteins with prosthetic groups (e.g. heme or chlorophyll) or labelled with fluorescent dyes.

### Clear native PAGE

CN-PAGE (commonly referred to as Native PAGE) separates acidic water-soluble and membrane proteins in a polyacrylamide gradient gel. It uses no charged dye so the electrophoretic mobility of proteins in CN-PAGE (in contrast to the charge shift technique BN-PAGE) is related to the intrinsic charge of the proteins. The migration distance depends on the protein charge, its size and the pore size of the gel. In many cases this method has lower resolution than BN-PAGE, but CN-PAGE offers advantages whenever Coomassie dye would interfere with further analytical techniques, for example it has been described as a very efficient microscale separation technique for FRET analyses. CN-PAGE is also milder than BN-PAGE so it can retain labile supramolecular assemblies of membrane protein complexes that are dissociated under the conditions of BN-PAGE.