PolyacrylamideGel is a separation matrix used in electrophoresis of biomolecules, such as proteinsor DNAfragments. Traditional DNA sequencingtechniques 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 immunologyand protein analysis, often used to separate different proteins or isomersof the same protein into separate bands. These can be transferred onto a nitrocelluloseor PVDFmembrane to be probed with antibodies and corresponding markers, such as in a western blot. PAGE is the often-used acronymfor polyacrylamide gel electrophoresis, where electrophoresis means applying an electric field to monitor the movement of particles through the polyacrylamide gel.
resolving gelsare 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
polymerizeuntil the ammonium persulfatehas 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
acrylamideis 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
* [http://www.gel-electrophoresis.com/gels/articles/polyacrylamide-gels/ Polyacrylamide Gels]
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Look at other dictionaries:
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polyacrylamide gel — noun : hydrated polyacrylamide that is used especially to provide a medium for the suspension of a substance to be subjected to gel electrophoresis … Useful english dictionary
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