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Information on Polyacrylamide Gels used in the molecular biology laboratory.
Polyacrylamide Gels
A Polyacrylamide Gel is a separation matrix used in electrophoresis of biomolecules, such as proteins or DNA fragments. Traditional DNA sequencing techniques such as Maxam-Gilbert or Sanger methods used polyacrylamide gels to separate DNA fragments differing by a single base-pair in length so the sequence could be read. Most modern DNA separation methods now use agarose gels, except for particularly small DNA fragments. It is currently most often used in the field of immunology and protein analysis, often used to separate different proteins or isomers of the same protein into separate bands. These can be transferred onto a nitrocellulose or PVDF membrane to be probed with antibodies and corresponding markers, such as in a western blot. The acryonym for polyacrylamide gel electrophoresis is often abbreviated to PAGE, where electrophoresis means applying an electric field to monitor the movement of particles through the polyacrylamide gel. What is Polyacrylamide? Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%. Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Because oxygen inhibits the polymerization process, they must be poured between glass plates (or cylinders). Advantages of Polyacrylamide Gels- Polyacrylamide forms gels with pores of a much more controlled and uniform size than does agarose. Consequently, polyacrylamide gels can be used to separate molecules that differ in size by as little as 2% of their molecular weight.
- The range of pore sizes possible is also much broader.
- Acrylamide is particularly superior when very small pore sizes are needed. It can be used to effectively separate small molecules (i.e. DNA oligonucleotides of 100 bases or less) that tend to run right through agarose gels.
- Another advantage of acrylamide as a gel matrix is that it is much stronger than agarose, producing gels that do not tear as nearly as easily.
- It is also possible to load larger quantities of material onto acrylamide gels and (because of the relative purity of the gel ingredients) to recover the material in a very pure (often still biologically active) form.
Disadvantages of Polyacrylamide Gels- Polyacrylamide gels are difficult to prepare when compared with agarose gels and the risk associated with their preparation is much greater.
- When unpolymerized, polyacrylamide acts as a cumulative neurotoxin. This means that it can cause serious neurological damage, and that the damage caused by exposure is compounded by each subsequent exposure. It is most dangerous in its powder form, when it is easily inhaled. Masks, gloves and safety glasses must always be worn when working with polyacrylamide powder. Whenever possible (and we will always do this in our laboratory), prepare polyacrylamide gels using pre-prepared liquid stock solutions.
Applications of Polyacrylamide Gels Polyacrylamide gels have a rather small range of separation, but very high resolving power. In the case of DNA, polyacrylamide is used for separating fragments of less than about 500 bp. However, under appropriate conditions, fragments of DNA differing is length by a single base pair are easily resolved. In contrast to agarose, polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins. Biosafety of PolyacrylamideAcrylamide is a potent neurotoxin and should be handled with care! Wear disposable gloves when handling solutions of acrylamide, and a mask when weighing out powder. Polyacrylamide is considered to be non-toxic, but polyacrylamide gels should also be handled with gloves due to the possible presence of free acrylamide.
Protocol for Preparing and Pouring Polyacrylamide GelsTypically resolving gels are made in 6%, 8%, 10%, 12% or 15%. Stacking gel (5%) is poured on top of the resolving gel and a gel comb (which forms the wells and defines the lanes where proteins, sample buffer and ladders will be placed) is inserted.
The percentage chosen depends on the size of the protein that one wishes to identify or probe in the sample. The smaller the known weight, the higher the percentage that should be used.
The mixtures below will not polymerize until the ammonium persulfate has been added, but if stored unpolymerized for long enough, the mixture may not polymerize correctly. Standard gel size is 3"x5"x0.2", and accounting for a small amount of leakage that generally occurs, each takes roughly 8mL of resolving and 2 mL of stacking gel. Types of Polyacrylamide Gels Polyacrylamide gels come in two main types in the molecular biology research laborotory, these are native gels and denaturing gels. Native Polyacrylamide Gels Native polyacrylamide gels also known as non-denaturing gels contain bis and polyacrylamide only. The catalysts ammonium persulfate (APS) and TEMED are added to speed up setting of the gel, however no denaturing chemicals such as SDS are added. Native polyacrylamide gels are often used to separate or recover proteins or other biological molecules from the gel in their native and biologically active form. Advantages of Native Gels: - more convenient to prepare
Disadvantages of Native Gels: - cannot separate proteins or even RNA based on their relative size as shape (secondary and tertiary structures) come into play.
Denaturing Polyacrylamide Gels Denaturing polyacrylamide gels are exactly like native gels with the exception that additional additives are added such as SDS in order to allow the separation of molecules by their relative size by keeping the molecules denatured or linearized. Formamide and urea is often used to denature DNA and RNA and allow their separation according to relative size by keeping the molecules single stranded. Heat is often used for nucleic acids to denature them as well. Proteins are kept in a denatured state by denaturing all secondary and tertiary structures using heat-activated sodium dodecyl sulfate (SDS), which is a detergent which binds to amino acids at a constant amount (every 3 aa) and disrupts interactions between side groups and disrupts all of the disulfide bonds that stabilize the higher order structure of proteins. SDS then coats the proteins thus preventing any of the higher order structure from re-forming. This means that all denatured proteins have roughly the same shape. SDS is added to the gel, as well as to the sample, to maintain all proteins in the denatured state. Because SDS is a (negatively charged) ionic detergent, when it coats proteins, it coats them with negative charges. Recall that proteins (in contrast to DNA) to not have a fixed charge-to-mass ratio (in DNA, there is one negative charge per nucleotide). This is why, in their native form, proteins cannot be separated by size using electrophoresis. But when proteins are coated with negatively charged SDS molecules, an artificial charge-to-mass uniformity is created-- the larger the protein molecule, the larger the number of SDS molecules needed to cover it. Under these circumstances (and given the shape uniformity also imposed by SDS-mediated denaturation), mixtures of proteins will migrate through a polyacrylamide gel with a speed based on their relative molecular weights. SDS, in effect, is able to artificially create for mixtures of proteins, the situation that exists naturally for mixtures of DNA fragments.
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