Staining is a critical step in the electrophoresis process. Commonly used protein dyes include Coomassie dyes, silver dyes, and fluorescent dyes. These staining reagents vary in sensitivity, ease of use, color, stability, versatility, and specificity. Ideal protein staining is characterized by high sensitivity, low detection limit, high dynamic range of quantitative accuracy, repeatability, and compatibility with post-electrophoretic protein identification systems.
Coomassie staining is the preferred method for simple, low-cost protein visualization. This protein staining can be detected simply by visual inspection and is relatively easy to use.
Coomassie dye is an organic dye able to detect proteins with lower detection levels of 8-10 ng. In acidic conditions, Coomassie dyes as anions bind nonspecifically to basic and hydrophobic residues of proteins, changing in color from a dull reddish-brown to intense blue. Since no chemical modification occurs, stained proteins can be recovered after complete decolorization for mass spectrometry and sequencing analysis. G-250 and R-250 are two types of Coomassie dyes. G-250 is characterized by a greenish color and reddish R-250 is less sensitive.
jetBLUE Protein Staining Solution is a ready-to-use colloidal Coomassie G-250 staining solution. COOMASSIE nano and 5X COOMASSIE nano, innovatively enhanced with proprietary nanotechnology, are new generation protein dyes that do not require destaining or washing steps.
|SDS-PAGE; mass spectrophotometry
|COOMASSIE nano 5X
|SDS-PAGE; mass spectrophotometry
|jetBLUE Protein Staining Solution
|PAGE; SDS-PAGE; isoelectric focusing; mass spectrophotometry
Silver staining is widely used in proteomic approaches and is the most sensitive colorimetric method for detecting total protein. Silver staining is a multi-step, time-consuming staining process, but its detection limit is 2 to 5 ng in per protein band. Silver staining may set the strict standard for ultimate detection sensitivity.
Silver staining depends on binding of silver ions from silver nitrate to various chemical groups (such as sulfhydryl and carboxyl moieties) of proteins. The silver ion is reduced to the metal silver which develops to a brownish black color. During silver staining process, sensitizers and enhancers are essential to control the specificity and efficiency of binding of silver ions to proteins and the effective conversion of bound silver to metallic silver.
There are a few noncovalent fluorescent dyes for electrophoretic applications, mainly the SYPRO dyes. Fluorescent staining can be a post-electrophoretic procedure. Alternatively, fluorescent dyes can be added to the running buffer, so that only a short destaining step is required after run. Most fluorescent staining mechanisms involve simple dye binding rather than chemical reactions that alter the functional groups of proteins. Stained proteins can be visualized using a standard 300-nm UV or blue-light transilluminator or other fluorescence imaging instruments.
Fluorescent dyes have several advantages over traditional protein stains. Depending on running conditions and fluorescent dyes, 1–10 ng may be detected, and the staining is reversible, more sensitive than Coomassie staining, and as sensitive as many silver staining techniques. Compared to typical silver staining, fluorescent staining is more straightforward and requires less hands-on time, and can be completed in less than 1 hour.
Coomassie Fluor Orange, SYPRO Orange, and SYPRO Red are chemically similar and bind to the detergent molecules surrounding proteins. SYPRO Ruby is a ruthenium metal chelate that binds to the basic amino acids in proteins by direct electrostatic interactions.
Electrophoresis is a laboratory technique used to separate mixtures of DNA, RNA, or protein molecules based on their size, mass, and electrical charge. Under the influence of an electric field, charged molecules migrate to separate in a matrix, such as agarose gel, polyacrylamide gel, or capillary tube.
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