Native Gel Electrophoresis Analysis Biology Essay

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Native Gel Electrophoresis is a technique used mainly in protein electrophoresis where the proteins are not denatured and therefore separated based on their charge-to-mass ratio.

The two main types of native gels used in protein electrophoresis are polyacrylamide gels and agarose gels.

Polyacrylamide gel electrophoresis (PAGE) is used for separating proteins ranging in size from 5 to 2,000 kiloDalton due to the uniform pore size provided by the polyacrylamide gel. Pore size is controlled by controlling the concentrations of acrylamide and bis-acrylamide powder used in creating a gel. Care must be used when creating this type of gel, as acrylamide is a potent neurotoxin in its liquid and powdered form. The other type of gel used is agarose gel. Agarose gels can also be used to separate native protein. They do not have a uniform pore size, but are optimal for electrophoresis of proteins that are larger than 200 kDalton.

Unlike SDS-PAGE type electrophoreses, Native gel electrophoresis does not use a charged denaturing agent. The molecules being separated (usually proteins) therefore differ inmolecular mass and intrinsic charge and experience different electrophoretic forces dependent on the ratio of the two. Since the proteins remain in the native state they may be visualised not only by general protein staining reagents but also by specific enzyme-linked staining.

SDS-PAGE (PolyAcrylamide Gel Electrophoresis)

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique widely used in biochemistry, forensics,genetics and molecular biology to separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight). SDS gel electrophoresis of samples have identical charge per unit mass due to binding of SDS results in fractionation by size.

The purpose of this method is to separate proteins according to their size, and no other physical feature. In order to understand how this works, we have to understand the two halves of the name: SDS andPAGE.

SDS

Since we are trying to separate many different protein molecules of a variety of shapes and sizes, we first want to get them to be linear so that the proteins no longer have any secondary, tertiary or quaternary structure (i.e. we want them to have the same linear shape). Consider two proteins that are each 500 amino acids long but one is shaped like a closed umbrella whle the other one looks like an open umbrella. If you tried to run down the street with both of these molecules under your arms, which one would be more likely to slow you down, even though they weigh exactly the same? This analogy helps point out that not only the mass but also the shape of an object will detrmine how well it can move through and environment. So we need a way to convert all proteins to the same shape - we use SDS.

Figure 1. This cartoon depicts what happens to a protein (pink line) when it is incubated with the denaturing detergent SDS. The top portion of the figure shows a protein with negative and positive charges due to the charged R-groups of the particular amino acids in the protein. The large H represents hydrophobic domains where nonpolar R-groups have collected in an attept to get away from the polar water that surrounds the protein. The bottom portion shows that SDS can break up hydrophobic areas and coat proteins with many negative charges which overwhelms any positive charge in the protein due to positively charged R-groups. The resulting protein has been denatured by SDS (reduced to its primary structure) and as a result has been lenearized.

SDS (sodium dodecyl sulfate) is a detergent (soap) that can dissolve hydrophobic molecules but also has a negative charge (sulfATE) attached to it. Therefore, if a cell is incubated with SDS, the membranes will be dissolved and the proteins will be soluablized by the detergent, plus all the proteins will be covered with many negative charges. So a protein that started out like the one shown in the top part of figure 1 will be converted into the one shown in the bottom part of figure 1. The end result has two important features: 1) all proteins contain only primary structure and 2) all proteins have a large negative charge which means they will all migrate towards the positve pole when placed in an electric field. Now we are ready to focus on the second half - PAGE.

 

PAGE

If the proteins are denatured and put into an electric field, they will all move towards the positive pole at the same rate, with no separation by size. So we need to put the proteins into an environment that will allow different sized proteins to move at different rates. The environment of choice is polyacrylamide, which is a polymer of acrylamide monomers. When this polymer is formed, it turns into a gel and we will use electricity to pull the proteins through the gel so the entire process is called polyacrylamide gel electrophoresis (PAGE). A polyacrylamide gel is not solid but is made of a laberynth of tunnels through a meshwork of fibers (figure 2).

Figure 2. This cartoon shows a slab of polyacrylamide (dark gray) with tunnels (different sized red rings with shading to depict depth) exposed on the edge. Notice that there are many different sizes of tunnels scattered randomly throughout the gel.

Figure 3. This is a top view of two selected tunnels (only two are shown for clarity of the diagram). These tunnels extend all the way through the gel, but they meander through the geland do not go in straight lines. Notice the difference in diameter of the two tunnels.

Now we are ready to apply the mixture of denatured proteins to the gel and turn on the current (figure 4). If all the proteins enter the gel at the same time and have the same force pulling them towards the other end, which ones will be able to move through the gel faster? Think of the gel as a tiny forrest with many branches and twigs througout the forrest but they form tunnels of different sizes. If we let children and adults run through this forrest at the same time, who will be able to get through faster? The children of course. Why? Because of their small size, they are more easily able to move through the forrest. Likewize, small molecules can manuver through the polyacrylamide forrest faster than big molecules.

Figure 4. Cartoon showing a mixutre of denatured proteins (pink lines of differen lengths) begining their journey through a polyacrylamide gel (gray slab with tunnels). An electric filed is established with the positive pole (red plus) at the far end and the negative pole (black minus) at the closer end. Since all the proteins have strong negative charges, they will all move in the direction the arrow is pointing (run to red).

You have to remember that when we work with proteins, we work with many copies of each kind of protein. As a result, the collection of proteins of any given size tend to move through the gel at the same rate, even if they do not take exactly the same tunnels to get through. Back to our analogy of the forrest... If we were in a hot air ballon above the forrest and watched 100 children, 100 teenagers, and 100 large adults running through the forrest, we would see collection (or band) of children moving quickly, a band of teenagers moving slower, and a third band made of adults plodding their way through the forrest. Likewize, proteins tend to move through a gel in bunches, or bands, since there are so many copies of each protein and they are al the same size. When running an SDS-PAGE, we never let the proteins electrophorese (run) so long that they actually reach the other side of the gel. We turn off the current and then stain the proteins (normally they are colorless and thus invisible) and see how far they moved through the gel. Figure shows a cartoon gel and figre 6 shows a one real. Notice that the actual bands are equal in size, but the proteins within each band are of different sizes.

Figure 5. This shows a top view of an SDS PAGE after the current has been on for a while (positive pole at the bottom) and then turned off. The gel (gray box) has five numbered lanes where five different samples of proteins (many copies of each kind of protein) were applied to the gel. (Lane 1, molecular weight standards of known sizes; Lane 2, a mixture of three proteins of different sizes with a being the biggest and c being the smallest protein; Lane 3, protein a by itself; Lane 4, protein b by itself; Lane 5 protein c by itself.) Notice that each group of the three proteins migrated the same distance in the gel whether they were with other proteins (lane 2) or not (lanes 3-5). The molecular weight standards are used to measure the relative sizes of the unknow proteins (a, b, and c).

Figure 6. This photo shows a variety of different proteins being separated on a gel. This particular image is showing a serial dilution of the same protein sample to indicate how little protein is needed (16 picograms = 16 . 10 -12 grams) in order to be detected. 

This image was taken from a home page operated by Hitachi Software (http://www.hitachi-soft.com/hitsoft/gs/fmbio/feb.htm)

There is a caveot to this method that you must always keep in mind. SDS-PAGE separates proteins based on their primary structure of size but not amino acid sequence. Therefore, if we had many copies of two different proteins that were both 500 amino acids long, they would travel together through the gel in a mixed band. As a result, we would not be able to use SDS-PAGE to separate these two proteins from each other.

Chapter 4: Electrophoresis - Introduction

Figure 4.1 Hoefer SE 400 Sturdier Electrophoresis units

Electrophoresis may be the main technique for molecular separation in today's cell biology laboratory. Because it is such a powerful technique, and yet reasonably easy and inexpensive, it has become commonplace. In spite of the many physical arrangments for the apparatus, and regardless of the medium through which molecules are allowed to migrate, all electrophoretic separations depend upon the charge distribution of the molecules being separated. 1

Electrophoresis can be one dimensional (i.e. one plane of separation) or two dimensional. One dimensional electrophoresis is used for most routine protein and nucleic acid separations. Two dimensional separation of proteins is used for finger printing , and when properly constructed can be extremely accurate in resolving all of the proteins present within a cell (greater than 1,500).

The support medium for electrophoresis can be formed into a gel within a tube or it can be layered into flat sheets. The tubes are used for easy one dimensional separations (nearly anyone can make their own apparatus from inexpensive materials found in any lab), while the sheets have a larger surface area and are better for two- dimensional separations. Figure 4.1 shows a typical slab electrophoresis unit.

When the detergent SDS (sodium dodecyl sulfate) 2 is used with proteins, all of the proteins become negatively charged by their attachment to the SDS anions. When separated on a polyacrylamide gel, the procedure is abbreviated as SDS--PAGE (for Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis). The technique has become a standard means for molecular weight determination.

Polyacrylamide gels are formed from the polymerization of two compounds, acrylamide and N,N-methylene- bis-acrylamide (Bis, for short). Bis is a cross-linking agent for the gels. The polymerization is initiated by the addition of ammonium persulfate along with either -dimethyl amino-propionitrile (DMAP) or N,N,N,N,- tetramethylethylenediamine (TEMED). The gels are neutral, hydrophillic, three-dimensional networks of long hydrocarbons crosslinked by methylene groups.

The separation of molecules within a gel is determined by the relative size of the pores formed within the gel. The pore size of a gel is determined by two factors, the total amount of acrylamide present (designated as %T) and the amount of cross-linker (%C). As the total amount of acrylamide increases, the pore size decreases. With cross- linking, 5%C gives the smallest pore size. Any increase or decrease in %C increases the pore size. Gels are designated as percent solutions and will have two necessary parameters. The total acrylamide is given as a % (w/v) of the acrylamide plus the bis-acrylamide. Thus, a 7 1/2 %T would indicate that there is a total of 7.5 gms of acrylamide and bis per 100 ml of gel. A gel designated as 7.5%T:5%C would have a total of 7.5% (w/v) acrylamide + bis, and the bis would be 5% of the total (with pure acrylamide composing the remaining 2.5%).

Proteins with molecular weights ranging from 10,000 to 1,000,000 may be separated with 7 1/2% acrylamide gels, while proteins with higher molecular weights require lower acrylamide gel concentrations. Conversely, gels up to 30% have been used to separate small polypeptides. The higher the gel concentration, the smaller the pore size of the gel and the better it will be able to separate smaller molecules. The percent gel to use depends on the molecular weight of the protein to be separated. Use 5% gels for proteins ranging from 60,000 to 200,000 daltons, 10% gels for a range of 16,000 to 70,000 daltons and 15% gels for a range of 12,000 to 45,000 daltons. 3

Cationic vs anionic systems

In electrophoresis, proteins are separated on the basis of charge, and the charge of a protein can be either + or -- , depending upon the pH of the buffer. In normal operation, a column of gel is partitioned into three sections, known as the Separating or Running Gel, the Stacking Gel and the Sample Gel. The sample gel may be eliminated and the sample introduced via a dense non-convective medium such as sucrose. Electrodes are attached to the ends of the column and an electric current passed through the partitioned gels. If the electrodes are arranged in such a way that the upper bath is -- (cathode), while the lower bath is + (anode), and -- anions are allowed to flow toward the anode, the system is known as an anionic system. Flow in the opposite direction, with + cations flowing to the cathode is a cationic system.

Tube vs Slab Systems

Figure 4.2 Electrophoretic separations of proteins

Two basic approaches have been used in the design of electrophoresis protocols. One, column electrophoresis, uses tubular gels formed in glass tubes, while the other, slab gel electrophoresis, uses flat gels formed between two plates of glass. Tube gels have an advantage in that the movement of molecules through the gels is less prone to lateral movement and thus there is a slightly improved resolution of the bands, particularly for proteins. It is also more economical, since it is relatively easy to construct homemade systems from materials on hand. However, slab gels have the advantage of allowing for two dimensional analysis, and of running multiple samples simultaneously in the same gel.

Slab gels are designed with multiple lanes set up such that samples run in parallel. The size and number of the lanes can be varied and, since the samples run in the same medium, there is less likelihood of sample variation due to minor changes in the gel structure. Slab gels are unquestionably the the technique of choice for any blot analyses and for autoradiographic analysis. Consequently, for laboratories performing routine nucleic acid analyses, and those employing antigenic controls, slab gels have become standard. The availability of reasonably priced commercial slab gel units has increased the use of slab gel systems, and the use of tube gels is becoming rare.

The theory and operation of slab gel electrophoresis is identical to tube gel electrophoresis. Which system is used depends more on the experience of the investigator than on any other factor, and the availability of equipment.

Figure 4.2 presents a typical protein separation pattern.

Continuous vs discontinuous gel systems

Figure 4.3 Schematic diagram of electrophoresis

The original use of gels as separating media involved using a single gel with a uniform pH throughout. Molecules were separated on the basis of their mobility through a single gel matrix. This system has only occasional use in today's laboratory. It has been replaced with discontinous, 4 multiple gel systems. In multiple gel systems, a separating gel is augmented with a stacking gel and an optional sample gel. These gels can have different concentrations of the same support media, or may be completely different agents. The key difference is how the molecules separate when they enter the separating gel. The proteins in the sample gel will concentrate into a small zone in the stacking gel before entering the separating gel. The zone within the stacking gel can range in thickness from a few microns to a full millimeter. As the proteins are stacked in concentrated bands, they continue to migrate into the separating gel in concentrated narrow bands. The bands then are separated from each other on a discontinuous (i.e. disc ) pH gel.5

Once the protein bands enter the separating gel, separation of the bands is enhanced by ions passing through the gel column in pairs. Each ioin in the pair has the same charge polarity as the protein (usually negative), but differ in charge magnitude. One ion will have a much greater charge magnitude than the proteins, while the other has a lesser charge magnitude than the proteins. The ion having a greater charge will move faster and is thus the leading ion, while the ion with the lesser charge will be the trailing ion. When an anionic system is employed, the Cl¯ and glycinate (glycine as its acid derivative) ions are derived from the reservoir buffer (Tris-Glycine). The leading ion is usually Cl¯ glycinate is the trailing ion. A schematic of this anionic system is shown in Figure 4.3. Chloride ions enter the separating gel first and rapidly move down the gel, followed by the proteins and then the glycinate ions. The glycinate ions overtake the proteins and ultimately establish a uniform linear voltage gradient within the gel. The proteins then sort themselves within this gradient according to their charge and size.

Agarose Gels

Figure 4.4 Agarose separation of cDNA

While acrylamide gels have become the standard for protein analysis, they are less suitable for extremely high molecular weight nucleic acids (above 200,000 daltons). In order to properly separate these large molecules, the acrylamide concentration needs to be reduced to a level where it remains liquid.

The gels can be formed, however, by the addition of agarose, a naturally linear polysaccharide, to the low concentration of acrylamide. With the addition of agarose, acrylamide concentrations of 0.5% can be used and molecular weights of up to 3.5 x 10 daltons can be separated. This is particularly useful for the separation of large sequences of DNA. Consequently, agarose-acrylamide gels are used extensively in today's genetic laboratories for the determination of gene maps. This chapter will concentrate on the separation of proteins, but Figure 4.4 demonstrates the separation of DNA fragments on an agarose gel.

Electrophoresis is a process to migrate ions in an electric field. In biochemistry, electrophoresis is commonly used to separate charged protein or nucleic acid molecules according to their size, shape and charge density.

Electrophoresis can be one dimensional or two dimensional. One dimensional electrophoresis is used for most protein and nucleic acid separations. Two dimensional separation of proteins is used in finger printing which is extremely accurate.

There are basically two types of electrophoresis, namely native gel electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE.

Native Gel Electrophoresis is a technique used mainly to separate proteins based on their charge to mass ratio where the proteins are not denatured. Native Gel Electrophoresis are further divide into two types, Polyacrylamide gel electrophoresis (PAGE) and agarose gel electrophoresis.

PAGE is suitable to separate protein molecules with size between 5 to 2000 kilo Dalton because the polyacrylamide gel has uniform pore size. The pore size is determined by two factors, the total amount of acrylamide present and the amount of cross-linker.

Agarose gel electrophoresis is suitable to separate molecules larger than 200 kilo Dalton, such as nucleic acids molecules. The pore size of agarose gel is not uniform.

The pH of the gel is high which is usually around pH 9 so that nearly all molecules have net -ve charges and move toward the positive electrode when the current is switched on. In this case, it is an anionic system where the electrodes are arranged in a way that the upper bath is -- (cathode), while the lower bath is + (anode). The -vely charged anions are allowed to flow toward the anode under certain duration of time.

Molecules of similar size and charge move as a band through the gel in where the mobility of smaller moecules is greater than the larger molecules with the same charge density.

After electrophoresis for a certain time, the separated bands may be visualized by general protein staining reagent and also by specific enzyme-linked staining or by radioactive labelling.

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique widely used to separate proteins according to their  molecular weight.

First step in SDS-PAGE is to incubate proteins in SDS. SDS (sodium dodecyl sulfate) is a detergent that is used to denature proteins and the proteins will be covered with many negative charges.

After denaturation, the protein now is linear, where no secondary or tertiary or quaternary structure present.

Next, the proteins are allow to move at different rates in a process called PAGE as mentioned before. The smaller molecules will move faster than the larger molecules in the gel. SDS-PAGE separates proteins based on their primary structure of size and not amino acid sequence. Therefore, if there are many copies of two different proteins that were both 600 amino acids long, they would travel together through the gel in a mixed band.Consequently, SDS-PAGE cannot separate these two proteins from each other. However, the technique has become a standard means for molecular weight determination.

ELECTROPHORESIS

In Biochemistry, gel electrophoresis is the process in which molecules such as proteins, DNA, or RNA fragments move in an electrical field with a velocity proportional to its overall charge density, size, and shape. There are basically two types of gel electrophoresis, namely native gel electrophoresis and sodium dodecyl sulphate polyacrylamide gel electrophoresis, SDS-PAGE.

Native gel electrophoresis is a technique used mainly to separate proteins based on their charge to mass ratio where the proteins are not denatured. Native gel electrophoresis is further divide into two types, polyacrylamide gel electrophoresis (PAGE) and agarose gel electrophoresis.

Agarose gel has a smaller resolving power as compared with polyacrylamide gel but a greater range of separation, that is from 200 bp to more than 50000 bp by using standard gels and electrophoresis equipment. The pore size of agarose gel is not uniform. On the other hand, polyacrylamide gel has a resolving power in the range of about 5 to 1000 bp. The polyacrylamide gel has uniform pore size. The pore size is determined by two factors, the total amount of acrylamide present and the amount of cross-linker. Polyacrylamide gel is much more difficult to handle than agarose gels.

The pH of the gel is high enough which is usually around pH 9 so that nearly all molecules have net negative charges and move toward the positive electrode when the current is switched on. In this case, it is an anionic system where the electrodes are arranged in a way that the upper bath is cathode, while the lower bath is anode. The negatively charged anions are allowed to flow toward the anode under certain duration of time. The electric current will force the molecules through a gel. For molecules with a relatively homogeneous composition such as nucleic acids having constant shape and charge density, the velocity is depends on size. Molecules of similar size and charge move as a band through the gel in where smaller molecules will move faster through than the larger molecules with the same charge density and thus migrate farther in a specific time. In gel electrophoresis, the sample molecules are not to be electrophoreses so long until they reach the other side of the gel, but at a specific time.

After electrophoresis, the separated bands may be visualized by general protein staining reagent or by radioactive labeling or by Western blotting. The gel may be soaking in a solution of a stain that binds tightly to proteins for example Coomassie Brilliant Blue R-250 or silver stain to visualize the separated proteins. The sizes of the various fragments can be determined by comparing their electrophoretic mobilities to the mobilities of fragments of known size. However, if the proteins in a sample are radioactive, the gel can be dried and then clamped over a sheet of X-ray film. The film will developed after some time and form an autoradiograph to be studied. The positions of the radioactive components are shown by the dark region on the film. On the other hand, if the protein consists of an antibody, immunoblotting or Western blotting is used to detect the specific protein on a gel in the presence of many other proteins. The samples contain less than a nanogram of protein can be separated and detected by gel electrophoresis depends on the dimensions of the gel and the visualization technique used.

SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis, is a technique widely used to separate proteins according to their  molecular weight. In SDS-PAGE, the proteins are to be denatured.

First step in SDS-PAGE is to incubate proteins in SDS. SDS, sodium dodecyl sulfate is a detergent that is used to denature secondary and tertiary and quaternary structures of protein and applies a negative charge to each protein in proportion to its mass. After denaturation, the protein now is linear, where only primary structure present. The SDS-treated proteins have similar shapes and mass to charge ratios.

Next, the proteins are allow to move at different rates in a process called PAGE as mentioned before. Since the SDS-treated proteins have similar shapes and mass to charge ratios, the rate at which they move towards the positive pole is the same, they also move in bunches as they are of same size. Therefore, we need to put the proteins into an environment that will allow different sized proteins to move at different rates such as polyacrylamide gel. The electricity is used to pull the proteins through the gel by polyacrylamide gel electrophoresis, PAGE.

The smaller molecules will move faster than the larger molecules in the gel. SDS-PAGE separates proteins based on their primary structure of size and not amino acid sequence. However, SDS-PAGE cannot separate two proteins of similar molecular weight from each other. Following electrophoresis, the gel may be stained to visualize the separated proteins. After staining, different proteins will appear as distinct bands within the gel. It is common to run molecular weight size markers of known molecular weight in a separate lane in the gel, in order to calibrate the gel and determine the weight of unknown proteins by comparing the distance travelled relative to the marker.

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