Electrophoresis


The June issue of Cold Spring Harbor Protocols includes an early preview of CSHL Press’ forthcoming RNA: A Laboratory Manual. Protocols covering basic RNA techniques are now available, including methods for purification of RNA by by SDS Solubilization and Phenol Extraction and by Using TRIzol (TRI Reagent), Ethanol Precipitation of RNA and the Use of Carriers, Preparation of Cytoplasmic and Nuclear RNA from Tissue Culture Cells, Removal of Ribosomal Subunits (and rRNA) from Cytoplasmic Extracts before Solubilization with SDS and Deproteinization, Removal of DNA from RNA, Nondenaturing Agarose Gel Electrophoresis of RNA and Polyacrylamide Gel Electrophoresis of RNA.

The last two on that list cover gel electrophoresis, two of the most important and frequently used techniques in RNA analysis. Electrophoresis is used for RNA detection, quantification, purification by size and quality assessment. Gels are involved in a wide variety of methods including northern blotting, primer extension, footprinting and analyzing processing reactions. The two most common types of gels are polyacrylamide and agarose. Polyacrylamide gels are used in most applications and are appropriate for RNAs smaller than approximately 600 nucleotides (agarose gels are preferred for larger RNAs). Polyacrylamide Gel Electrophoresis of RNA describes how to prepare, load and run polyacrylamide gels for RNA analysis. The is featured in the June issue, and as one of our featured articles, the full-text version is available to subscribers and non-subscribers alike.

This set is just a small sampling of the manual’s contents, basic techniques from an early chapter. The full table of contents can be seen here.

April’s issue of Cold Spring Harbor Protocols includes instructions for Rapid Coomassie Blue Staining of Protein Gels. This method is an adaptation of the conventional Coomassie staining protocol described in Staining Proteins in Gels with Coomassie Blue. Coomassie Brilliant Blue R250 (CBR-250) is the most commonly used dye for visualizing proteins because of its relatively high sensitivity. The modified method speeds up the destaining process for faster results with increased sensitivity and is compatible with mass-spectrometry-based methods for identifying proteins. Other methods for staining proteins can also be found in Cold Spring Harbor Protocols, including the Zinc/Imidazole Procedure for Visualization of Proteins in Gels by Negative Staining, and Staining Proteins in Gels with Silver Nitrate. Silver Nitrate’s sensitivity is in the low-nanogram range, which is 50-100 times more sensitive than classical Coomassie Blue staining, ~10 times better than colloidal Coomassie Blue staining, and at least twice as sensitive as the zinc/imidazole negative staining method.

Our second featured article for the May issue of Cold Spring Harbor Protocols is Systematic Monitoring of Protein Complex Composition and Abundance by Blue-Native PAGE, written by Harvey Millar and colleagues from the University of Western Australia. The article describes multiple experimental approaches using polyacrylamide gel electrophoresis (PAGE). Blue-native PAGE (BN-PAGE) allows a range of protein complexes to be visualized. When combined with sodium dodecyl sulfate PAGE (SDS-PAGE), the procedure can resolve the complexes and their subunits by their molecular weight. In conjunction with differential in-gel electrophoresis (DIGE), BN-PAGE can be used to quantify changes in protein complex abundance or subunit composition between different samples. A detailed methodology is provided for BN-PAGE, SDS-PAGE, and DIGE, and like all of our featured articles, it is freely accessible to subscribers and non-subscribers alike.