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Additional recommended knowledge
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.
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 extremely toxic when ingested, and absorbs into the skin very easily. Once the acrylamide is polymerized it is no longer absorbable, but care still should be taken when disposing of the gel
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Polyacrylamide_gel". A list of authors is available in Wikipedia.|