Gel Types for Specific Applications
SDS-PAGE and native PAGE — Bis-Tris, Tris-acetate, and Tris-HCl gels are available for PAGE applications. SDS-PAGE is the most common protein electrophoretic application. It separates complex protein mixtures by size, allowing estimation of the molecular weight of sample proteins. Bio-Rad’s Tris-HCl gels are made without SDS. By omitting SDS from the running buffer,the gels can be used to run proteins under nondenaturing conditions for subsequent analysis of their native conformation and activity. Native analysis does not provide accurate molecular weight information. Gels for SDS-PAGE and native PAGE protein separations have traditionally been formulated with Tris-HCl. The neutral-pH formulations of Bis-Tris and Tris-acetate gels significantly delay acrylamide hydrolysis and give these gels longer shelf life (12 months for Bis-Tris gels, 8 months for Tris-acetate gels) than conventional Laemmli Tris-HCl gels. By using optimized combinations of Criterion XT neutral-pH gel buffers and running buffers, all sizes of proteins can be separated into tight, well-resolved bands on a range of only two or three acrylamide percentages. Criterion XT gels can also be used for SDS-PAGE or native PAGE applications.
Small protein and polypeptide analysis — Tris-Tricine gels are ideal for separation of peptides and small proteins with molecular weight <10,000. Superior resolution is achieved by slowing the migration rate of the peptide-SDS complexes. This helps achieve separation from the faster-moving SDS micelles that interfere with peptide resolution in Tris-glycine buffer systems.
IEF — IEF gels separate proteins based on their net charge rather than molecular weight. IEF gels are cast with carrier ampholytes to create a pH gradient within the gel. Proteins migrate to their pI, the pH at which their net charge is zero. IEF gels contain no denaturing agents, allowing one-dimensional separation under native conditions.
Protease detection — Zymogram gels contain either gelatin or casein, which are substrates for various proteases. Samples are run under denaturing but nonreducing conditions, then allowed to renature and consume the substrate. Samples with proteolytic activity can be visualized as clear bands against a blue background after Coomassie Blue R-250 staining.
dsDNA separation — TBE gels are suitable for electrophoresis of nucleic acids from 50 to 2,000 base pairs; they are ideal for analysis of the purity of PCR products, standard dsDNA analysis, and RNase protection assays.
ssDNA and RNA separation — TBE-urea gels maintain denaturing conditions for analysis of single-stranded DNA and RNA. Nucleic acids between 60 and 200 bases are resolved as sharp, distinct bands. Applications include oligonucleotide purity analysis, RNase protection assays, and northern blotting.
Acrylamide Percentage (%T)
Proper acrylamide concentration is critical to the success of electrophoretic separation. Acrylamide concentrations that are too high can lead to exclusion of high molecular weight molecules from the gel; concentrations that are too low can decrease sieving. Use the protein and DNA migration charts (pages 188 190) to select a gel with a %T that will provide optimal resolution of your sample.
Single-percentage gels — Choose a single-percentage gel when your sample has a limited size range of molecules and your goal is to separate a single band from neighboring bands. In general, single-percentage gels will produce the greatest separation between bands with similar molecular weights. Single-percentage Tris-HCl, Bis-Tris, Tris-acetate, Tris-Tricine, and zymogram gels are cast with a 4% stacking gel to further sharpen protein bands before they enter the resolving gel.
Gradient gels — Choose a linear gradient gel if your sample contains a wide range of molecular weights. These gels allow both high and low molecular weight bands to be visualized on the same gel.
Crosslinker Percentage (%C)
Crosslinker percentage is the weight percentage of crosslinker (bis-acrylamide). Along with the total monomer concentration or %T (acrylamide), %C determines the pore size of the gel. Crosslinker percentage can be adjusted to optimize pore size in order to deliver the best separation and resolution for the molecule of interest. Bio-Rad precast gels have a %C optimized for the bestseparation of molecules for their applications. Refer to the table below for the crosslinker percentage in Bio-Rad precast gels.
