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Introduction

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).

Post-electrophoretic protein stains

Coomassie brilliant blue stain (CBB)

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.

Staining method

  1. Dissolve 0.025–0.10% (w/v dye) dye in an acidic alcohol formulation [30– 50% (v/v) methanol (or, less frequently, ethanol)) with 7–10% (v/v) acetic acid solution.
  2. Filer the solution with Whatman #1 paper for a stable formulation.
  3. Perform the fixation and staining in 10 gel volumes of solution.
  4. Destain with 7% acetic acid.

Strengths and limitations

Coomassie brilliant blue stain is easily available and is the most commonly used protein stain. However, staining and destaining require more time and reagents.

Silver staining

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.

Staining method

  1. After separation, fix the gel in fixation solution (50% methanol, 12% acetic acid, and 0.05% formalin) for 2 hours or overnight, followed by three washes with 35% ethanol for 20 minutes each.
  2. Sensitize gel for 2 minutes, followed by three washes in water for 5 minutes each.
  3. Stain the gel in silver nitrate solution (0.2% silver nitrate and 0.076% formalin) for 20 minutes, followed by two washes in water.
  4. Add the developer (6% sodium carbonate, 0.05% formalin, and 0.0004% sodium thiosulfate) and stop staining by leaving the gel for 5 minutes in stop solution (50% methanol and 12% acetic acid).

Strengths and limitations

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 stain

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.

Staining method

  1. After electrophoresis, incubate the gel in 0.2 M imidazole, 0.1% Sodium dodecyl sulfate SDS for 15 minutes.
  2. Discard the imidazole solution and incubate the gel in 0.3 M zinc sulfate for 30-45 seconds.
  3. Discard the developer and wash the gel for several times, with water, 1 minute per wash.
  4. Store the gel in 0.5% (w/v) sodium carbonate.

Strengths and limitations

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.

Fluorescent total protein stains

SYPRO ruby

SYPRO Ruby is a luminescent ruthenium metal chelate that interacts with the basic amino acids in proteins. It provides with the 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 of spots identification.

Staining method

  1. Fix the gel in 50% (v/v) methanol, 10% (v/v) acetic acid, and leave it for 30 minutes or overnight.
  2. Stain the gel with SYPRO ruby stain.
  3. Briefly destain the gel with 10% (v/v) methanol and 7% (v/v) acetic acid.
  4. Visualize under UV light or with blue light sources; the protein bands appear orange-red to the eye.

Strengths and limitations

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 stain

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.

Staining method

  1. Dilute the Nile red from a stock solution (0.4 mg/mL in dimethyl sulfoxide DMSO) 200-fold into the water to 2 mg/mL.
  2. Add a tenfold volume excess (e.g., 50 mL staining solution for a 5 mL mini gel) to a gel and thoroughly agitate.
  3. Briefly wash the gel with water after staining.
  4. The gels can be viewed under UV light or with green light sources; the protein bands appear pale red.

Strengths and limitations

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.

Epicocconone Stain

The epicocconone stain is a fluorescent stain which was isolated from a 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.

Staining method

  1. Fix the SDS–PAGE in 7.5% (v/v) acetic acid for 60 minutes.
  2. Wash the gel with water (2×30 min).
  3. Stain the gel for 1 hour in an aqueous solution into which an aliquot of the fluorophore stock solution is diluted.
  4. Incubate the gel in 0.05% (v/v) ammonia (3×10 min).
  5. Acquire the images with UV or blue or green visible light.

Strengths and limitations

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.

References

  1. Chevalier, F., (2010). Standard Dyes for Total Protein Staining in Gel-Based Proteomic Analysis. Materials (Basel), 3(10), 4784–4792.
  2. R. Sundaram., N. Balasubramaniyan., & Sundaram., P. (2012). Protein stains and applications. Methods Mol Biol, 869, 451-464.
  3. , H. T., (2009). Protein gel staining methods: an introduction and overview. Methods Enzymol., 463, 541-63.
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