Polyacrylamide gel

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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.

Resolving gels

Typically 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.

Recipes

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.

To make 10 ml of a 10% (resolving) polyacrylamide mixture:

dH20                            4.0 ml
30% acrylamide mix              3.3 ml
1.5M Tris pH8.8                 2.5 ml
10% SDS                          .1 ml
10% ammonium persulfate          .1 ml
TEMED                            .004 ml

To make 10 ml of a 5% (stacking) polyacrylamide mixture

dH20                            5.65 ml
30% acrylamide mix              1.65 ml
1.0M Tris pH6.8                 2.5 ml
10% SDS                          .1 ml
10% ammonium persulfate          .1 ml
TEMED                            .004 ml

Caveat: acrylamide is toxic when ingested and can be absorbed through the skin. Once the acrylamide is polymerized it is no longer absorbable, but care still should be taken when disposing of the gel

External links

it:Gel di poliacrilammide


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