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Gel Electrophoresis: Lambda Phage

Understand how to stain a gel.

Jul 28th, 2015

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Once protein bands have been separated by electrophoresis, they can be visualized using different methods of in-gel detection. Over the past several decades, demands for improved sensitivity for small sample sizes and compatibility with downstream applications and detection instrumentation have driven the development of several basic staining methods over the years. Each method has particular advantages and disadvantages, and an number of specific formulations of each type of method provide optimal performance for various situations. This article discusses the general principles of protein gel staining and describes several types of staining methods.

General principles of gel staining

The first step after performing denaturing polyacrylamide gel electrophoresis (SDS-PAGE) is to disassemble the gel cassette and place the thin (1 mm thick) polyacrylamide gel in a tray filled with water or buffer. The electrophoresed proteins exist as concentrated "bands" embedded within each lane of the porous polyacrylamide gel matrix. Typically, the proteins are still bound to anionic SDS detergent, and the entire gel matrix is saturated in a particular buffer.

To make the proteins visible, a protein-specific, dye-binding or color-producing chemical reaction must be performed on the proteins within the gel. Depending on the particular chemistry of the stain, various steps are necessary to hold the proteins in the matrix and to facilitate the necessary chemical reaction. All steps are done in solution, i.e., with the gel suspended in a tray filled with one liquid reagent or another.

Given the common constraints of this format, most staining methods involve some version of the same general incubation steps:

  • A water-wash to remove electrophoresis buffers from the gel matrix
  • An acid- or alcohol-wash to condition or fix the gel to limit diffusion of protein bands from the matrix
  • Treatment with the stain reagent to allow the dye or chemical to diffuse into the gel and bind (or react with) the proteins
  • Destaining to remove excess dye from the background gel matrix

Depending on the particular staining method, two or more of these functions can be accomplished with one step. For example, a dye reagent that is formulated in an acidic buffer can effectively fix and stain in one step. Conversely, certain functions require several steps. For example, silver staining requires both a stain-reagent step and a developer step to produce the colored reaction product.


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Jul 28th, 2015

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