Protein separation and identification is critical for proteome analysis and requires high resolution and powerful protein characterization after gel electrophoresis. To identify the proteins in the gel, colorimetric and fluorescence staining techniques are used.
The stains include anionic dyes (Coomassie brilliant blue), metal cations (imidazole-zinc), silver stain, fluorescent dyes, and radioactive probes. Specific staining methods could also be used to detect post-translational modifications such as glycosylation or phosphorylation.
The protein stain should exhibit high sensitivity (i.e., low detection limit), quantitative accuracy, reproducibility, ease of use, and compatibility with downstream protein analysis techniques like mass spectrometry (MS).
Coomassie brilliant blue stain is a disulfonated triphenylmethane dye that is used to stain protein bands bright blue. The stain binds with the protonated basic amino acids (lysine, arginine, and histidine) by electrostatic interactions and with the aromatic residues by hydrophobic interactions.
The CBB has a low affinity for the polyacrylamide, but it does penetrate the gel matrix, necessitating a destaining step. Coomassie stains are noncovalent, reversible, and do not interfere with downstream analysis techniques such as mass spectrometry of excised protein bands.
Coomassie brilliant blue stain is easily available and is the most commonly used protein stain. However, staining and destaining require more time and reagents.
The silver stain is the alternative colorimetric stain for increased detection sensitivity as compared to the Coomassie staining. It is generally considered as the standard for other “ultrasensitive” staining methods.
Silver staining protocols are categorized on the basis of the silvering agents and development conditions. Silver diamine complex in an alkaline environment is used in alkaline methods; acidic silver nitrate is used in acidic methods.
Silver staining is one of the most sensitive colorimetric methods used for the detection of total protein. The formulations and protocols are optimized and standardized, helping to minimize the effects of minor differences in day-to-day use.
Zinc staining is a negative stain that stains the polyacrylamide gel except proteins enabling their detection. Zinc ions coupled with imidazole precipitates in the gel matrix.
Zinc ion staining utilizes the ability of proteins and protein-SDS complexes to bind and sequester Zn2+ in the gel, causing the precipitation with opaque background, contrasting with transparent protein-SDS-Zn2+ zones.
The technique is compatible with mass spectrometry or microsequencing methods for downstream protein analysis and characterization.
The zinc stain is a sensitive dye for the detection of 0.25 ng of protein per band in a mini gel. The entire method takes 15 minutes.
Proteins could be easily recovered from the gel after staining. Overdevelopment of the stain can be problematic but can be rectified by incubating in 100 mM glycine to dissolve excess zinc imidazolate.
SYPRO Ruby is a luminescent ruthenium metal chelate that interacts with the basic amino acids in proteins. It provides a sensitivity close to that of silver staining and the properties of classical organic stains such as Coomassie blue.
The staining does not lead to any irreversible modification of amino acids, so satisfactory mass spectrometry compatibility is expected. It also allows a stable sequence coverage with a capacity for spots identification.
The stain could be profitably used for large-scale proteomic analysis. The staining procedure is simple and allows high-throughput and large-scale proteomic applications. However, the necessity of a fluorescent scanner makes it expensive.
Nile red is a phenoxazone dye that presents strong fluorescence upon transition from aqueous to hydrophobic environments like SDS micelles or protein–SDS complexes.
Nile red does not bind the SDS monomers; this property makes it a rapid, non-fixative total protein staining method for SDS gels.
Detection sensitivity of the stain is similar to that of Coomassie stains. Nile red-stained gels could be subsequently electroblotted with an excellent transfer efficiency. High-fluorescent background and photostability are the limitations of this staining technique.
The epicocconone stain is a fluorescent stain that was isolated from the fungus Epicoccum nigrum. It is an azaphilone that reacts with primary amines and NH3 to produce red fluorescent compounds which detect proteins in the gels.
The stain is a rapid and sensitive fluorescent total protein stain suitable for both protein gels and blots. It is highly compatible with mass spectrometry, Edman sequencing, and Ettan™ DIGE system.
It is also environmentally friendly and does not contain any heavy metals, allowing safe disposal after use. The staining method for protein identification in gel should be chosen based on its sensitivity and downstream analysis techniques.
While selecting the stain, the composition of the proteins of interest (presence/ absence of single amino acids), sample availability, and post-staining application of the gel/protein should be considered.
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