Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Methods for Staining Proteins in Gels
Protein Staining Methods Using Dyes

Protein staining in gels using Dyes is less sensitive than the silver complex Methods described above, but it is easier to perform in routine laboratory practice. The most widely used dye is Coomassie Brilliant Blue, although many other modifications of the method exist. Coomassie-stained protein bands can be photographed under fluorescent lighting on Type 665 Polaroid film (focal length 32, exposure 0.5 s) using a yellow filter [353].

1. Staining of PAGE gels with Coomassie R250 (0.1% w/v, Serva) in a mixture of 50% methanol and 12% acetic acid overnight with gentle agitation. Destaining with several changes of a 10% ethanol – 5% acetic acid solution. Photography through a narrow-band (550 nm) Interference filter (Baird Atomic, Bedford, MA) [253]. Following SDS-PAGE of Proteins tightly bound to DNA, two artifact bands (corresponding to molecular weights of 54∙10³ and 68∙10³) stainable with Coomassie and silver complexes were detected; the buffer contained mercaptoethanol [364].

2. Staining of polyacrylamide gels overnight with Coomassie R250 (0.2% w/v) in a 45% methanol – 10% acetic acid mixture, followed by destaining with a 10% methanol – 7% acetic acid solution [317]. Following Electrophoresis, agarose gels were rinsed with distilled Water, wrapped in filter paper, and incubated for 30 min at 37 °C. Staining became visible 15 min after Treatment with Coomassie R250 (0.1% w/v) in a 45% ethanol – 10% acetic acid mixture. Destaining was carried out with the same solvent mixture.

3. Detection of proteins and Polypeptides with Coomassie following isoelectric focusing [370]. Coomassie Brilliant Blue R250 cannot be used under standard conditions to detect proteins in PAGE after isoelectric focusing because carrier ampholytes (polyamino-polycarboxylic acids) react with the dye. To overcome these limitations, The Use of colloidal Suspensions of Coomassie R250 and G250 in perchloric [311] and trichloroacetic [26, 313] acids has been proposed. The Procedure outlined below yields excellent results.

Following isoelectric focusing, a 0.5 mm thick gel strip is immersed for 3 h in an aqueous 0.15% Coomassie G250 solution containing 40% methanol and 4% formaldehyde. Destaining is performed overnight with 10% methanol by replacing the washing solution several times. A faint blue Background remains, but it does not interfere with the assay. Photographs are taken under fluorescent lighting on a Type 665 Polaroid sheet with an orange filter, focal length, exposure 1/8 s.

The method is applicable to PAGE systems containing SDS and 8 M urea. Polypeptides and proteins (M 1665–68,000) with isoelectric points ranging from pH 2.2 to 10.2 were separated by isoelectric focusing in a pH 3.5–10.5 gradient. The detection sensitivity for the tridecapeptide of $\alpha$-melanocyte-stimulating hormone was 20 \mu g.

4. Staining with Coomassie during electrophoresis [379].

The method enables staining during electrophoresis in acidic gels and has been used primarily for the detection of Histones. Coomassie R250 (0.01%, w/v) is added to the cathode buffer, and electrophoresis is carried out in 0.9 M acetic acid (pH 2.5) at a load of 4 mA per gel tube and 100 V until the dye reaches the top of the gel. Destaining is performed for a short time (2–3 h) to reveal minor bands.

5. The following procedure was used to detect histones on PAGE.

Prior to silver staining, the gel is immersed for 3 h in 500 mL of a solution containing 1% amido black, 40% methanol, and 7% acetic acid. Destaining is carried out overnight in 4 L of a 32% ethanol–18% acetic acid mixture, followed by four washes with 1 L of water each [259].

Alternatively, the gel is gently agitated for 3 h (maximum) in a solution containing 0.05% 2,7-naphthalenedisulfonic acid (Eastman), 50% methanol, and 10% acetic acid. No destaining is required.