PAGE Gel Chemistries for Protein Separation
TGX (Tris/Glycine eXtended) PAGE Gels are based on a modification of the Laemmli system. These gels enable very fast run times without distortion and have a longer shelf life than standard Laemmli gels, retaining their performance and reproducibility for up to a year. This novel SDS-PAGE gel chemistry uses the same sample separation profiles as Laemmli gels and the same sample and running buffers. Precast TGX gels are available in a range of percentages, including gradient gels, with different well configurations and volumes in both mini and midi sizes.
TGX Stain-Free PAGE Gels are a recent innovation providing the ability to do quality control after both electrophoresis and blotting or when locating bands for spot cutting. With stain-free technology, a PAGE gel can be visualized immediately after electrophoresis to confirm that sample loading is in the right range and run quality is satisfactory with no artifacts such as smiling or vertical streaking; bands can be identified and excised for further analysis such as mass spectrometry. After blotting transfer, blots can be instantly visualized to assess the efficiency of transfer and the quality of the blot.
The ability to directly view the quality of a PAGE gel and a blot prior to further processing saves time and resources. Precast TGX and TGX Stain-Free Gels are available in the same wide range of percentages and well configurations in mini and midi formats.
Tris-HCl and Tris-acetate PAGE gels are formulated without SDS. This gives the flexibility of using a single type of gel for either separation of native or SDS-denatured proteins. Presence of SDS in the sample and running buffer creates denaturing conditions with separation comparable to Laemmli SDS-PAGE gels. Proteins separated in the absence of SDS (and usually also reducing agent) are used in protocols where proteins need to remain in a native configuration. Migration patterns under native conditions differ from those in denaturing gels, and molecular weight cannot be determined with accuracy.
Bis-Tris PAGE gels can also be used for the separation of either native or denatured proteins. This PAGE gel system has a discontinuous buffer system like Laemmli, but is run at a lower pH. Either MES or MOPS running buffers can be used with Bis-Tris gels. MOPS buffer is usually preferred for midsized proteins, whereas MES is used for smaller proteins. Due to the lower pH, Bis-Tris gels have a longer shelf life than standard Laemmli gels.
Tris/tricine PAGE gels are formulated to separate proteins and peptides with molecular weights of 10 kDa and below. The slower mobility of proteins in Tris/tricine gels than in Tris/glycine gels results in better separation of low molecular weight polypeptides away from SDS micelles running close to the migration front.
Specialized PAGE Gels for Protein Analysis
IEF PAGE gels are used to separate proteins by charge. In a pH gradient, proteins migrate until they have no net charge — when the pH equals the isoelectric point (pI) of the protein. An IEF PAGE gel can be used to separate native proteins or proteins with charged post-translational modifications (such as phosphorylation). Precast gels are available with both broad and narrow pH gradients. Additionally, for samples that have been electrophoresed in immobilized pH gradient (IPG) strips for 2-D electrophoresis, mini and midi precast gels are available with wells that accommodate the strips for the second-dimension electrophoresis.
Zymogram PAGE gels detect proteins that have proteases that can use gelatin or casein as a substrate. Proteins are separated on the gels under denaturing, nonreducing conditions. After electrophoresis, the PAGE gel is incubated in zymogram renaturing buffer. After renaturation of the proteins, the gel is then incubated in zymogram development buffer containing a cation cofactor required for protease activity. Proteases are usually identified as clear bands (where the casein or gelatin has been proteolysed) on a blue background after Coomassie staining.
PAGE Gel Chemistries for Nucleic Acid Separation
TBE PAGE gels are nondenaturing gels for the separation of nucleic acids. TBE gels are used for dsDNA analysis, to assess the purity of PCR products and for RNase protection assays. Nucleic acids from 50 to 2,000 bp to are efficiently separated on TBE gels. Bio-Rad has a range of precast TBE gels in mini and midi sizes.
TBE-urea PAGE gels are used for both RNA and ssDNA. A denaturing PAGE gel is used for determination of oligonucleotide purity, northern blotting and RNase protection assays. TBE-urea gels give sharp, tight bands with an optimal size range up to 200 bp. Precast gels are available in mini and midi formats.