Western Blot Gel Types

If you are studying protein phosphorylation or monitoring expression of different proteins within similar molecular weight ranges, getting efficient separation between targets becomes critical. The percentage composition and type of SDS-PAGE gel you use could influence electrophoresis and detection of samples.

Fixed Gels

A fixed or a straight SDS-PAGE gel consists of stacking (top) and resolving (bottom) acrylamide matrix components. The stacking component has a lower concentration of acrylamide, compared to the resolving component, which concentrates the sample before separation1. The resolving gel has a homogenous composition and uniform pore size to enable separation of proteins with similar molecular weights.

Gradient Gels

A gradient gel consists of a matrix with different concentrations of acrylamide. The pore sizes at the top of the gel are usually larger (lower concentration of acrylamide), with sizes gradually decreasing towards the bottom of the gel (higher concentration of acrylamide). Gradient gels are effective at separating proteins in a complex sample with a broad range of molecular weights. Depending upon molecular weight differences, gradient gels may be able to effectively separate phosphorylated and non- phosphorylated forms of a protein of interest.

Combination

For samples containing protein complexes, it is best to separate them on a gradient gel first and identify a narrower molecular weight range for the proteins of interest. The samples can then be electrophoresed on a fixed gel to get better resolution between similar molecular weight proteins.

Other considerations

Both fixed and gradient gels can be poured in the lab or alternatively purchased pre-cast. The number of wells, well volume, and type of buffering system (phosphate-buffered saline or tris-buffered saline) to be used are all important considerations for effective protein separation.

References

  1. Overview of Protein Electrophoresis. Thermo Fisher Scientific.
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