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Impacts of Polyacrylamide Concentration on Protein Experiments

Polyacrylamide is a common reagent in the molecular biology lab as it is used in the formation of gels used to study proteins in techniques such as Western blotting and polyacrylamide gel electrophoresis (PAGE). Many scientists take advantage of these techniques for their research, but may not be aware of the impact that the polyacrylamide concentration of the gel being used can have on an experiment.

Polyacrylamide gels are formed by the polymerization of acrylamide and bis-acrylamide (bis). On its own, acrylamide forms a linear chain. Bis is used to crosslink chains of acrylamide polymers. This polymerization and crosslinking is catalyzed by free radicals derived from the reaction between ammonium persulfate and TEMED. The mixture of acrylamide, bis, ammonium persulfate, and TEMED forms a polyacrylamide matrix that can be used for distinguishing proteins by size and/or shape.

The chemical properties of acrylamide and bis make polyacrylamide gels ideal for use in studying proteins, as they will not react with proteins. Because of this, proteins can be analyzed in polyacrylamide gels in their native state or be manipulated by researchers to be analyzed in a denatured state.

It’s important for researchers to choose a gel based on the ability of their protein of interest to be resolved on the gel. Changing the amount of acrylamide and bis-acrylamide in the gel composition changes its density, and the density of a gel impacts the pore size, or the size of the gaps between the molecules in the gel. The higher the density of polyacrylamide in a gel, the smaller the pore size. Gels with a higher density can be used to distinguish between molecules as small as 4-10 kDa, while lower density gels can differentiate between larger proteins of upwards of 200 kDa. Gels are commonly used at a polyacrylamide percentage of between 4% and 20% due to the fragility of gels with higher or lower concentrations.

Stacking gels, in which the top of the gel has a lower concentration of polyacrylamide than the bottom of the gel, can improve the resolution of a PAGE or Western blotting experiment. The top of a stacking gel is known as the stacking portion, and the bottom as the separating or resolving portion. Using a stacking gel, all proteins migrate from the wells through the stacking portion of the gel to concentrate at the interface, and enter the resolving portion of the gel simultaneously. In contrast, when using a single density gel, proteins migrate from the well directly into the resolving gel sequentially, and this can give rise to smearing on the final blot.

For researchers interested in investigating multiple proteins of different sizes from the same sample, there are two options: either to run multiple gels of different densities to identify each protein separately, or to use a gradient gel that allows for proteins of disparate sizes to be identified from the same blot. Gradient gels have a low polyacrylamide concentration at the top of the gel that gradually increases towards the bottom of the gel. Common gradient gels have polyacrylamide at 4% polyacrylamide at the top of the gel and 20% at the bottom. Large proteins of up to 200 kDa can be observed at the top of the gel, and proteins as small as 5 kDa can be found at the bottom.

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Aliyah Weinstein

Posted by Aliyah Weinstein

Aliyah is a scientist and science communicator based in the Boston area. She has a PhD in immunology and currently works in the nonprofit sector and as a freelance science writer.