The Protein Man's Blog | A Discussion of Protein Research

How to visualize proteins after electrophoresis

In-gel visualization of proteins separated by SDS-PAGE or by 2-D gel electrophoresis is achieved by staining gels with dye, metals ions or fluorescent stains. Different staining method are preferred depending upon broad range binding of stain to proteins, the sensitivity to detect low abundant proteins and downstream application desired for a separated protein like mass spectrometry. Sometimes two staining methods are carried out as single staining method cannot achieve everything desired by a researcher. Various staining method for in-gel visualization of proteins are as follows.

Coomassie Brilliant Blue staining

The Coomassie dyes R-250 and G-250 bind to proteins stoichiometrically through their sulfonic acid groups. The interactions between dye and protein are Van der Waals and ionic. The sulfonic acid groups interact with positive amine groups. Therefore coomassie dye binds to wide range of proteins. The most commonly used dye R-250 can detect protein as low as 0.1 µg. Coomassie Brilliant Blue involves staining and destaining step as the gel also turns blue. Coomassie Brilliant blue is compatible with mass spectrometry analysis. Though widely used for staining gels to view proteins, its not a method of choice if higher sensitivity is desired. For analytical purposes other methods like silver staining and fluorescent staining are preferred over classical Coomassie Brilliant Blue staining.

Colloidal Coomassie G-250 staining

In colloidal Coomassie staining G-250 dye is used instead of R-250 due to its colloidal properties. G-250 forms colloidal particals in high alcoholic solutions containing strong acids and high concentration of salts ^{1, 2}. Formation of colloidal G-250 particals reduces free dye in solution and thus the dye does not penetrate and stain gel. This step enhances the sensitivity of staining proteins and removes background staining of gels. With colloidal coomassie staining detection limit of 1-10 ng of protein in gel is achieved³.

Silver staining

It offers high sensitivity of less than 1 ng of protein⁴. The silver ions bind to the protein and are reduced further with a developing solution to give an image of protein bands. Silver staining involves various steps like protein fixation, sensitization, silver impregnation and image development. Based on solution used there are two methods of silver staining: silver nitrate and silver-ammonia complex method. Silver staining protocols compatible with downstream application like mass spectrometry are available. The downside of silver staining is that it has limited dynamic range and thus do not interact uniformly with all proteins. In addition it involves use of hazardous chemicals.

Fluorescent staining

Fluorescent staining uniformly stains protein, is rapid and offers sensitivity equal to that attained by silver staining method. It involves no destaining step and is compatible with mass spectrometry and microsequencing. Commercially fluorescent stains are available under trade name SYPRO® Protein Stains to stain 1D or 2D gels to visualize bands. The sensitivity of SYPRO® Ruby Gel Stain is 0.25 ng.

Stain free detection of proteins in gel

In stain free method for visualizing proteins, 2, 2, 2-Trichloroethnaol is added to the polyacrylamide solution before casting a gel⁵. The principle behind stain free method is that the tryptophan amino acids in protein undergo an ultraviolet light-induced reaction with trihalocompounds to produce fluorescence in visible range (300 nm). This method is fast as one can see bands on transilluminator within 5 minutes and it avoids use of stains.

References

1. Neuhoff, V. et al (1988). Electrophoresis 9, 255-262
2. Candiano, G. et al (2004). Electrophoresis 25, 1327-1333
3. Kang, D. et al (2002). Bull. Korean Chem. Soc. 11, 1511-1512
4. Chevallet, M. et al (2006). Nat Protoc.1(4): 1852-1858
5. Ladner, C. L. et al (20014). Anal Biochem. 2004 Mar 1, 326(1):13-20