3 Protein Gel Stains and When You Should Use Them

After the protein bands have been separated by electrophoresis, researchers perform a dye-binding or color-producing chemical reaction to visualize the proteins embedded within the pores of the polyacrylamide gel matrix. Some of the most popular in-gel detection methods include staining with Coomassie dye, metallic silver, and zinc.

Considering the number of protein gel stains available, how do you choose one for your particular application? Here is a guide that can help you achieve your goal.

Coomassie Dye Stains

Despite the wide range of protein stains available today, most researchers still use Coomassie dye for in-gel protein detection. This may be largely due to the fact that it can effectively stain proteins in a relatively short period of time (usually within the hour) and does not require methanol or acetic acid for staining.  

Coomassie dye binds to basic and hydrophobic residues of proteins and changes from dull reddish-brown to intense blue under acidic buffer conditions. Like other staining methods, it can detect some proteins better than others.

Here are some of the other advantages of using Coomassie dye:

• It can detect as few as 8-10 nanograms per band for some proteins and 25 nanograms per band for most proteins.
• It involves a single, ready-to-use reagent.
• It does not permanently modify the target proteins. Thus, the excised protein bands can be completely destained and the proteins recovered for analysis by mass spectrometry or sequencing.

Coomassie dyes come in two variants (R-250 and G-250) and are commonly used as the basis of stains for detection of proteins in gel electrophoresis and Bradford-type assay reagents for protein quantitation.

Silver Stains

In this method, metallic silver is deposited onto the surface of a gel at the location of protein bands. The silver ions then react with certain protein functional groups (particularly with carboxylic acid groups Asp and Glu, imidazole, sulfhydryls, and amines) resulting in brown-black color.

While commercially available silver staining kits are robust and easy to use (they can be used to detect less than 0.5 nanograms of protein in typical gels), their accuracy and efficiency is largely dependent on reagent quality, incubation times, and the thickness of the gel. In addition, the method uses glutaraldehyde or formaldehyde as enhancers which can cause chemical crosslinking of the proteins in the gel matrix. This may limit the efficiency of destaining and recovery of proteins for downstream applications such as mass spectrometry. Therefore, when using silver staining in mass spectrometry, you should carefully consider which is more important - optimization of sensitivity or protein-recoverability.

Due to its high sensitivity, silver stains are most ideal for applications involving low protein levels.

Zinc Stains

While all the other staining methods stain the proteins, this technique stains all the areas of the gel except those that contain proteins. Basically, the zinc ions interact with imidazole which then precipitates in the gel matrix except in areas where SDS-saturated protein occurs. The precipitate (which is milky white in color) turns the background opaque while the protein bands remain clear and virtually unstained. The whole usually takes less than 15 minutes and the gel can be photographed by viewing the gel over a dark background.

Advantages of the zinc staining method:

• The method is as sensitive as typical silver stains and can detect less than 1 ng of protein.
• It requires no fixation steps.
• The stain is easily removed. Thus, the proteins can be easily recovered.
• It is compatible with mass spectrometry or Western blotting.

Zinc stains are ideally used prior to the following:

• Protein recovery for antibody generation or immunological detection.
• Excision of gel pieces for protein digestion and analysis by mass spectrometry.
• Electrophoretic transfer to membrane for Western blotting.

It is also ideal for quick purity checks before proceeding with purification or transfer for Western blotting.

Image source: m01229