Imaging Coomassie-stained gels using NIR fluorescence and white light

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What is Coomassie?

Coomassie is a blue stain used in proteomics-related studies to detect proteins during electrophoresis, SDS-PAGE, and Bradford assays. In Western blot analysis, Coomassie is used as a loading control and an anionic pre-antibody stain. Coomassie binds non-specifically to almost all proteins. It is also the most common method of in-gel protein detection, whose popularity can be attributed to its characteristics of being efficient, quick, and affordable. 

In this blog post, we will cover how to use NIR fluorescent to image coomassie-stained gels.

How to image Coomassie stained gels using white light or using NIR fluorescence

To begin, soak the gels in the dye. To reduce background and make the bands easier to visualize, elude the extra stain with a solvent; this step is called destaining. Destaining can take as little as 10 minutes, to overnight to produce bands with clear background.

Doing these steps allows for the visualization of proteins as blue bands on a clear background. Shown below is a serial dilution of bovine serum albumin (BSA) run on an SDS-PAGE gel and stained with a colloidal Coomassie stain. The same gel was imaged 3 ways.

The white light images were captured after placing the gel on a white light transilluminator table using white light on the Azure 300 Imager (Figure 1) and new chemiSOLO (Figure 2). The NIR fluorescence image was captured using the 700 channel (excitation wavelength 685nm) on the Sapphire Biomolecular Imager (Figure 3). On all images, the protein bands are easily visible down to the lowest amount loaded: 90 ng.

Coomassie-stained gel imaged on the Azure 300 Imager on a white light transilluminator
Figure 1. Coomassie-stained gel imaged on the Azure 300 Imager on a white light transilluminator
Coomassie-stained gel imaged on the Azure chemiSOLO
Figure 2. Coomassie-stained gel imaged on the Azure chemiSOLO using white light
Coomassie-stained gel imaged in the 700 channel (NIR fluorescence) on the Azure Sapphire Biomolecular Imager
Figure 3. Coomassie-stained gel imaged in the 700 channel (NIR fluorescence) on the Sapphire Biomolecular Imager

NIR fluorescence imaging of Coomassie-stained gels

About 15 years ago, it was reported that Coomassie blue-bound protein fluoresces in the near infrared3. Since that time, infrared fluorescent imaging of Coomassie stained gels has not become routine, perhaps limited by the availability of instruments able to carry out near-infrared fluorescence imaging3. Butt et al conducted a systematic study comparing the sensitivity and linear dynamic range of seven commercial Coomassie stains and seven published stain formulations when imaged using NIR fluorescence3.

The Coomassie stains were also compared to Sypro Ruby, which, though expensive, is a popular fluorescent stain due to its high sensitivity, high dynamic range, low interprotein variability, and mass spec compatibility. Butt et al found that some Coomassie stain formulations exceeded Sypro Ruby with respect to sensitivity and linear dynamic range3.

In examining 2D gels of mouse brain extract, Butt et al found more proteins were detected in gels stained with Sypro Ruby. The authors hypothesized the difference was due to attenuation of Sypro Ruby fluorescence in high-abundance spots, which may allow longer imaging to detect low abundance spots without signal saturation. This would suggest a tradeoff between sensitivity and quantitative accuracy when deciding between Sypro Ruby and Coomassie to stain a 2D gel3.

The sensitivity and linear dynamic range observed in this study suggests Coomassie may be an attractive, less expensive option for fluorescent staining of protein gels in some situations.

White light imaging of Coomassie-stained gels

In a Coomassie-stained gel, the protein bands appear blue in a clear background may be examined by eye on the bench or on a light box. For record keeping, the gel may be imaged photographed with white light. The sensitivity of traditional Coomassie staining depends on the specific protein being assessed. Detection limit per protein band is about 30 to 100 ng of protein and can approach 10 ng2, 3. The sensitivity of traditional Coomassie staining is about 10 ng2.

In addition to R-250 (the original Coomassie Brilliant Blue ) and G-250 (the demethylated derivative of Coomassie Brilliant Blue ), colloidal Coomassie stains are available in which the dye is present in micelles1. Colloidal stains result in lower background than traditional Coomassie stains because the micelles are too large to enter the polyacrylamide gel matrix1. Colloidal Coomassie blue staining can provide higher sensitivity, as low as 1 ng2. depending on the formulation3,4, 5.

Scientist using pliers to place gel inside Azure chemiSOLO interface
The new Azure chemiSOLO is used to image Coomassie-stained gels, chemiluminescent Western blots, and more.

Why Coomassie staining is so popular for SDS-PAGE gels

Coomassie blue is one of the most commonly used dyes for staining proteins in SDS-PAGE gels. A variety of Coomassie stain formulations can be purchased commercially or mixed in the lab. The staining protocol typically involves incubating the gel in staining solution until bands are visible, followed by destaining to remove background.

The entire staining process with Coomassie can take from a few hours to overnight. Destaining reduces sensitivity and is essential with traditional Coomassie staining because high background is common. Coomassie dyes bind protein through electrostatic and hydrophobic interactions1.

Advantages of using Coomassie

Advantages of Coomassie staining include simplicity and affordability. It is also compatibile with downstream mass spectrometry analysis. 

Disadvantages of using Coomassie

Disadvantages include low sensitivity compared to other staining methods. Staining intensity can depend in part on amino acid composition of the proteins, which complicates the use of Coomassie for relative quantitation of different proteins1.

Looking for better ways to visualize your proteins? Azure Biosystems offers a range of imagers capable of imaging Coomassie-stained gels under white light (epi or trans-illumination). The new Azure chemiSOLO is able to easily and quickly image chemiluminescent Western blots without additional software downloads. It’s the first personal Western blot imager of its kind on the market! Get a quote for chemiSOLO by clicking here.

Frequently Asked Questions about coomassie

Coomassie blue stain binds proteins non-covalently so it is compatible with downstream analyses such as mass spectrometry which is frequently used to identify the protein in a spot or band from a gel. Multiple formulations of Coomassie stain are commercially available and a variety of staining techniques have been published. Read more about Visible Gel Imaging here.

When used in acidic conditions, Coomassie will bind to the hydrophobic, basic residues in proteins. Its color will change from a dull copper, red/brown hue to an intense shade of blue.

There are a number of issues that can arise during a Western blot transfer.  Many times it can be resolved by adjusting the transfer time, temperature or simply remaking the buffers. More troubleshooting tips are available in this blog post: Trouble-free Transfers.

SOURCES

  1. Smejkal GB. The Coomassie chronicles: past, present and future perspectives in polyacrylamide gel staining. Expert Rev Proteomics. 2004;1(4):381-387.
  2. Butt RH and Coorssen JR. Coomassie Blue as a near-infrared fluorescent stain: a systematic comparison with Sypro Ruby for in-gel protein detection. Mol Cell Proteomics. 2013;12(12):3834-3850.
  3. Butt RH and Coorssen JR. Coomassie Blue as a near-infrared fluorescent stain: a systematic comparison with Sypro Ruby for in-gel protein detection. Mol Cell Proteomics. 2013;12(12):3834-3850.
  4. Neuhoff V, Arold N, Taube D, Ehrhardt W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis. 1988;9(6):255-262.

  5. Candiano G, Bruschi M, Musante L, et al. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis. 2004;25(9):1327-1333.

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