This is the last section of the 2-D gel electrophoresis. Herein we will discuss about the different techniques used for visualization and the staining of the proteins in the 2-D PAGE.

Protein visualization and staining

A large number of methods are available for protein detection. The choice of stain depends mainly on the experimental workflow. There is a wide variation in the detection efficiency, accuracy to quantify protein amount, compatibility with the mass spectrometry, complexity of procedures and cost. Visualization of 2-DE is somewhat different than SDS-PAGE. Proteins are resolved as a spot instead of discrete band. However, the addition of a second dimension to the gel does not any way hinder visualization, instead it adds a sharpness and defined boundaries to the define spot.

There are 4 major categories of protein staining for 2-DE. They are: organic dye staining, silver staining (classical and mass spectrometry- compatible), Fluorescent staining and Phosphoprotein stains.

  1. Organic dye staining: This includes coomassie blue R and G type stains. Chemically, colloidal coomassie blue G- 250 stain (CBB) consists of triphenylmethane, which has two methyl groups more than brilliant blue. G, variant refers that the blue stains has a slight green tint. While the 250 refers to the purity. CCB has a limited protein detection ranged from 8- 50 ng within the spot. The dye complexes with basic amino acids, such as tyrosine, histidine, arginine, and lysine. The formation of protein- dye complexes stabilizes the negatively charged anionic form of the dye producing a clear protein spot with faint background color. That is because the property of colloidal nature of the stain prevents it to penetrate to the gel. On binding to protein, a negative charge of the dye will dominate the protein – dye complexes. This feature can be used to separate proteins and protein complexes under denaturating condition using polyacrylamide gel electrophoresis in a technique called Blue – native gel. A major advantage of CBB is the compatibility with mass spectrometry.
  2. Silver staining: Among the various protein detection methods following 2-DE, silver staining has gained wide popularity because of its sensitivity. Ammoniacal silver stain provides a high sensitivity (2-4 ng) over CBB staining [30 – 100 times more sensitive]. The high sensitivity of silver staining makes it more susceptible to interference with other reagents even the purity of water, deionized water should be used. Proteins bind silver ions, which can be reduced under appropriate conditions to build up a visible image made of finely divided silver metal. Silver- nitrate has better procedure and speed than silver- ammonia (ammoniacal).  However, basic protein are less efficiently stained than acidic ones. Moreover, they do not work properly below 19–20°C, except when the water used for making the solutions from steps is warmed at 20–25°C or above prior to use. A major drawback of silver staining that it is not compatible with mass spectrometry. This is because proteins within the gel to be analyzed should remain in its unmodified status. Kits for MS- compatible silver staining are available
  3. Fluorescent staining: Highly sensitive fluorescent stains include different families like SYBRO, CyDye, and Deep purple. The flurophors have a wide liner range of detection which facilitates a powerful and accurate quantitative evaluation of the sport intensity. Their sensitivities are slightly higher or comparable to silver staining but not as high as radiolabelling. Deep purple stain can detect protein spot as 0.5 ng. It is compatible to mass spectrometry. This stain can be used with ultraviolet transillumination (365 nm), light boxes (400-500 nm), laser- based scanners (457, 488, or 532 nm excitation), and CCD cameras. Sybro Ruby stain is high sensitive stains. It is a ruthenium- based metal chelate fluorescent stain. The ruby stain has been shown to detect 20% more protein compared to silver stain. It has a liner dynamic ranges that covers three order magnitudes. Also its merit over silver stain in its short processing time and need no fixation step prior to staining. CyDye provides a multicolor dye staining. Cy2, Cy3 and Cy5, which exhibit different excitation and emission spectra, can be used in differential gel electrophoresis (DIGE). Providing an excellent quantitative interpretation for protein spot analysis. Its rather expensive and need to be analyzed in an over layed computerized method
  4. Phosphoprotein and glycoprotein stains: A colorimetric stain to specifically detect proteins separated by polyacrylamide gel electrophoresis (SDS-PAGE) or 2-DE. Protein of interest that is phosphorylated at serine and/or threonine residues can be detected using these stains. Staining is achieved by first hydrolyzing the phosphoprotein-phosphoester linkage using 0.5N NaOH in the presence of calcium ions. The gel containing the newly formed insoluble calcium phosphate is then treated with ammonium molybdate in dilute nitric acid. The resulting insoluble nitrophospho-molybdate complex is finally stained with a solution of the basic dye, methyl green. In glycosylated proteins, separated proteins is treated with a periodate solution, which oxidizes cis-diol sugar groups in glycoproteins. The resulting aldehyde groups are detected through the formation of Schiff-base bonds with a reagent that produces magenta bands (PAS staining). Several commercially available stains for both modifications such as (Pro-Q Diamond, Gelcode, Pro-Q Emerald or AMRESCO) are now available. These types of stains are of great significance for detecting post- translational modification and understanding disease-related proteins.

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This is all for 2D-PAGE.

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Please read other posts by The Biotech Notes:

Real time PCR

Flow cytometer

Immunoprecipitation- P1

References:

Magdeldin et al (2012). Two-Dimensional Polyacrylamide Gel Electrophoresis – A Practical Perspective, Gel Electrophoresis – Principles and Basics, Sameh Magdeldin, IntechOpen, DOI: 10.5772/36816.