Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Protein Staining Methods in Gels
Protein Staining with Silver Complexes

Warning! Silver-ammine complexes are explosive and must not be stored. Spent solutions containing these complexes should be treated with HCl to decompose them and diluted with Water before disposal.

To recover silver, add a saturated NaCl solution, separate the liquid by filtration and discard it, and collect the AgCl precipitate [408].

Following silver staining, results can be continuously recorded by photographing them on Polaroid film (3X5) or negative film (from which prints can be made later). A method using direct-copy film (Kodak X-Omat) under UV illumination for 1–1.5 s, followed by Processing with Kodak developer and fixer, has been described. This Procedure is not applicable to Coomassie-stained samples [125].

8.19.2.1. Photodevelopment of Proteins in PAGE.

Procedure A [253]. Visual detection of proteins takes less than <50 min.

1. After Electrophoresis, fix the protein for 10 min in a solution containing 50% methanol and 12% acetic acid.

2. Wash the gel twice with a 10% ethanol – 5% acetic acid mixture.

3. Soak the gel in a 0.5% potassium hexacyanoferrate(II) solution for 5 min with gentle agitation.

4. Decant the solution and wash the gel three times with water (20 s per wash).

5. Add a solution containing 0.2 g of silver nitrate, 0.2 g of ammonium nitrate, 0.5 mL of 37% formaldehyde (Fisher Scientific), and 0.06 mg of benzotriazole in 100 mL of water.

6. Irradiate the gel for 20 min in a lightbox using a 160 W fluorescent lamp, model T-12 (Aristo Grid Products), with an emission intensity equivalent to that of a 500 W tungsten light source.

7. Decant the silver nitrate solution, and without washing the gel, add 200 mL of a 3% sodium carbonate solution containing 37% formaldehyde and 0.6 mg benzotriazole/L. Continue irradiation with gentle agitation. After 1 min, a brown precipitate forms, and the sodium carbonate solution is replaced with fresh solution. The process is terminated once the desired staining intensity is reached.

8. Staining can be stopped at any moment by removing the sodium carbonate solution and soaking the gel (for 5 min) in 1% acetic acid.

9. Wash the gel with water before storage.

Procedure B [256]. This method allows the detection of proteins and Nucleic Acids within 10 min.

1. Fix the gel by incubating it for 5 min in a methanol – acetic acid – water mixture (100:20:80) containing 4 g of citric acid and 0.4 g of NaCl.

2. Rinse the gel with 200 mL of deionized water to remove NaCl.

3. Place the gel in 200 mL of a methanol – acetic acid – water mixture (100:20:80) containing 4 g of silver nitrate, and illuminate as described in Procedure A.

Take a series of photographs during development (for quantitative analysis, every 10 min).

4. Stop the staining by placing the gel in the dark. Sensitivity is 0.5 ng of protein. DNA exhibits negative staining.

8.19.2.2. Polyacrylamide gels.

Procedure [242].

Silver staining Reagents. Complex solution. Mix (1:5) a 30% methylamine solution (commercial grade) with an NaOH solution (0.36%, w/v) and add ~10 mL of this solution to 4 mL of 20% (w/v) AgNO₃ until a brown precipitate forms and the solution clears. Bring the total volume to 100 mL with water. Observe safety precautions; see Section 8.19.2.

Developer. Add 15 mL of a solution containing 1% (w/v) citric acid and 1.5 mL of 40% formaldehyde (commercial grade) to 3 L of water.

Destaining reagents.

A. Dissolve 11.1 g of NaCl and 11.1 g of CuSO₄ in 285 ml of water, and add a 25% ammonia solution until a precipitate forms and the dark blue solution clears (final volume ∼300 ml).

B. Dissolve 44 g of sodium thiosulfate (pentahydrate) in 85 ml of water (final volume ∼100 ml).

C. Kodak clearing agent: 20 g in 800 ml of water.

Staining procedure.

1. Soak the PAGE gel (0.8–3.0 mm thick) after electrophoresis overnight in a solution of 50% methanol + 10% acetic acid (100 ml).

2. Wash at 60 °C for 10 and 20 min, changing the water several times (200 ml).

3. Incubate for 30 min at 60 °C in a 0.1% (w/v) formaldehyde solution (200 ml) prepared by adding 2.5 ml of 40% formaldehyde to 1 L of water.

4. Cool to room Temperature for 10 min in water (200 ml).

5. Shake for 10 min at room temperature in the silver complex solution (200 ml).

6. Decant the complex solution and rapidly rinse the gel with two volumes of water and developer (100 ml each).

7. Keep the gel in the developer for 30 min, changing the solution every 5 min, until the Background darkens due to the silver mirror reaction. Protein bands gradually (5–25 min) become intensely stained.

8. Wash the gel for 10 and 20 min with two changes of cold water and soak overnight in a fresh portion of water.

Destaining Procedure. Mix reagents A and B in a 3:1 ratio and dilute with an equal volume of water. Incubate the gel in this solution (100 ml) for 1–4 min until the background turns yellow. Quickly rinse with water and immerse for 30 min in reagent C (100 ml). Wash with three volumes of water (incubating for 10, 20, and 30 min, respectively).

Note. The method is based on earlier works [241, 244]. It provides high sensitivity (0.01 ng BSA/mm²) due to the low formaldehyde concentration and an intensive staining procedure followed by destaining. Using two-dimensional electrophoresis combined with this method, more than 200 Peptides from 30 µl of urine and over 150 from a single fingerprint were separated and detected. See also [243] for the staining of human saliva proteins using silver complexes.

8.19.2.3. Large-pore PAGE [295, 244]. Large-pore gels are used for the electrophoretic fractionation of macromolecules with molecular weights > 106. Agarose gels darken upon Treatment with silver complexes; therefore, the staining procedure described above cannot be applied to them [35]. Below is the staining procedure for large-pore gels prepared from 2.55% polyacrylamide with 2.75% methylenebisacrylamide cross-linking.

Procedure.

1. After electrophoretic protein Separation, soak the gel for 1 h in a 37% formaldehyde (Fluka) – ethanol – water mixture (15:27:63) and then leave it overnight in a mixture of the same components at a different concentration (1:25:75).

2. Shake the gel at 60 °C in a methanol – acetic acid – water mixture (5:7:88) for at least 4 h, changing the solution every 20–30 min.

3. Shake the gel at 60 °C for 15 min with a freshly prepared solution of 4% (w/v) paraformaldehyde (Merck) in 1.43% (w/v) sodium cacodylate (Fluka), pH 7.3.

4. Wash the gel at 60 °C with distilled water for at least 3 h, changing the water every 20–30 min.

5. Place the gel in the silver complex solution (section 8.19.2.2) for 10 min at room temperature.

6. Rinse with water for 2–3 min.

7. Develop with a freshly prepared aqueous solution of citric acid (0.005%, w/v; Merck) and formaldehyde (0.019%, w/v; Merck), constantly changing the solution until dark protein bands appear.

8. Stop the staining by adding methanol (5%).

8.19.2.4. Agarose gels after isoelectric focusing. The 10-min silver complex staining method is 20 times more sensitive than Coomassie R250 staining and can be used after dye staining [211].

Fixation and drying. Following isoelectric focusing, proteins in the gels are fixed by immersion for 10 min in a solution of 10% trichloroacetic acid—1% sulfosalicylic acid. Wash for 10 min with 95% ethanol. Dry in a stream of warm air.

Autoradiography. For low-energy isotopes, Ultrofilm [³Н] LKB 2208-190 film is used, processed with LX24 developer and AL4 fixer for Kodak X-ray film.

Coomassie staining. Immerse the gel for 5 min in a solution containing 35% ethanol, 10% acetic acid, and 0.6% Coomassie R250. Destain for 10 min with a 35% ethanol—10% acetic acid mixture.

Silver complex staining.

1. After fixation, place the gel for 5 min in a solution containing 50% methanol, 12% trichloroacetic acid, 2% СuСl₂, and 1% ZnCl₂, and dry it.

2. Immerse the gel in a (1:2) mixture of 0.01% КМnО₄ and Harris's hematoxylin histological stain (Chroma 2Е 0565). Stain composition: 10 ml ethanol + 1 g hematoxylin + 20 g potassium ammonium phosphate + 0.5 g yellow mercuric oxide.

3. Rinse the gel in distilled water (for a few seconds); the orange color fades and turns pinkish-lilac.

4. Preparation of Solutions. A. 8 g of anhydrous sodium carbonate in 100 ml of distilled water (prepared daily). B. Dissolve in 80 ml of water (in the specified order) 0.28 g of ammonium nitrate, 0.2 g of silver nitrate, 1 g of silicotungstic acid (SiО₂∙12WО₃∙26H₂О), and 0.73 ml of 35% formaldehyde. Bring the volume to 100 ml (can be stored for several months at room temperature). Mix equal volumes of solutions A and B immediately before use and immerse the gel in this mixture. Within 1—3 min, the protein bands turn reddish-brown and then black.

5. Stop the reaction by rinsing the gel in distilled water for a few seconds.

The detection limit of the method is 0.045 µg of protein per gel band. See also [380] for protein staining with silver complexes in agarose gels.

8.19.2.5. Polyacrylamide and agarose gels after glutaraldehyde treatment. The method is based on single-step silver reduction following protein treatment with glutaraldehyde. Washing steps are omitted in this description.

Reagents. Glutaraldehyde solution (Merk-Schuchardt). Ammoniacal silver solution (0.2% w/v): mix an NaOH solution (0.36 g in 21 ml of water), 1.4 ml of NH₄OH (28% w/v), and 1 ml of silver nitrate (20% w/v), and bring the total volume to 100 ml with water. Take appropriate safety precautions; see Section 8.19.2.

Polyacrylamide gels. After electrophoresis, fix the protein for 30 min in a 45% methanol—10% acetic acid solution. Wash with water to remove organic Solvents. Place the gel in 200 ml of a 1% (w/v) glutaraldehyde solution and incubate with vigorous shaking for 15 h at room temperature.

Strict adherence to the glutaraldehyde concentration is crucial; a 1% solution is suitable for 120×140 mm gels, and a 0.5% solution for 30×45 mm gels at a thickness of 0.75—1.5 mm.

Transfer the gel to the silver complex solution for 30 min. Vigorous agitation prevents silver precipitation on the gel. Background staining can be selectively removed within a few minutes by treatment with 10% acetic acid.

Wash the gel with water (or phosphate-buffered saline, pH 7.2, if acetic acid was used). Photograph. Store the gel sheets in plastic containers. The sensitivity is 0.03 ng/mm² for BSA and 0.5 ng/mm² for Ovalbumin, which is 100-fold higher than with Coomassie staining.

Agarose gels. Following Immunoelectrophoresis, moisten the agarose gel with distilled water or saline, blot with filter paper, and dry for 15 min. Transfer to 100 ml of the silver complex solution for 5—40 min. Remove background staining as described above. Use vigorous agitation at all stages.

8.19.2.6. Silver complex staining with subsequent toning [23]. The intensity of stained protein bands can be increased 7- to 10-fold using the procedure described below. Densitometric evaluation demonstrated that the linear relationship between staining intensity and protein concentration is maintained in the range of 0.05—1.7 pmol/mm².

Reagents.

A. 5% (w/v) iron(II) chloride solution.

B. 3% (w/v) oxalic acid.

C. 3.5% (w/v) potassium ferricyanide solution.

These solutions can be stored for several months at room temperature.

Toning bath. Mix 10 ml of each of reagents A, B, and C, and add 70 ml of water. Prepare immediately before use. The solution must be brown; if a green-blue color develops, it is unsuitable for use.

Procedure. Polyacrylamide gels are stained with a silver complex [269], rinsed in running tap water for at least 30 min, and then transferred to a toning solution for 0.5–3 min, whereupon protein bands turn blue. Following a 10-min wash in running tap water, the staining is stabilized by soaking for 10 min in a 20% methanol – 5% acetic acid mixture; the gels are stored in heat-sealed polyethylene bags. Quantitative evaluation is performed densitometrically at 550 nm following silver staining, and spectrophotometrically at 660 nm after toning, using a Gilford Model 250 spectrophotometer. Gels are photographed on Kodak Panatomic film.

8.19.2.7. Double staining with Coomassie and silver complex [176]. Pre-treatment with Coomassie combined with simultaneous formaldehyde fixation enhances staining intensity and facilitates the identification of bands on the gel.

Fixation and Coomassie treatment.

1. Following electrophoresis, the PAGE gel is immersed for 1 h in a formaldehyde – acetic acid – ethanol – water (5:10:25:60) solution containing Coomassie R250 (0.05% w/v), and then for 3 h or overnight in a solution of the same components at a ratio of 0.5:10:25:64 containing 0.05% Coomassie R250.

2. The staining solution is decanted, the gel is rinsed with an acetic acid – isopropanol – water (10:25:65) mixture, and subsequently with a mixture of the same solvents in a 10:10:80 ratio until the background is clear.

Silver staining (modified procedure) [252].

1. Following fixation, wash the gel with a 10% aqueous ethanol solution to remove acetic acid.

2. Rinse for 30 min with a mixture of 4% paraformaldehyde and 1.43% (w/v) sodium cacodylate (adjusted to pH 7.6 with HCl).

3. Wash with 10% ethanol 4 times for 5 min each.

4. Incubate for 30 min in a copper nitrate – silver nitrate solution (dissolve 3.5 g of silver nitrate in 100 ml of water and add 1.5 ml of a 0.5% (w/v) copper solution containing 4 ml of pyridine and 8 ml of absolute ethanol).

5. Wash for 5 min in a freshly prepared complex solution: mix 30 ml of a 19.4% (w/v) silver nitrate solution and 22.2 ml of an alkaline-ammoniacal aqueous-ethanol solution (100 ml of 0.36% aqueous NaOH + 45 ml of concentrated ammonia + 55 ml of 20% ethanol).

6. Treat twice for 1 min with the reducing solution: 2.5 ml of 10% formaldehyde (prepared by diluting commercial formaldehyde with water, 1:10) + 6 ml of 1% citric acid + 100 ml of ethanol; bring the total volume to 1 L with water. Then rinse several times with the second reducing solution: 5 ml of 10% formaldehyde + 5 ml of 1% citric acid + 100 ml of ethanol; bring the total volume to 1 L with water; the treatment is continued until brown and black protein bands appear on the electropherogram.

7. Stop development by immersing the gel in 1% acetic acid. Gels can be stored in this solution for at least 2 months; alternatively, the plates can be vacuum-dried at 100 °C on filter paper. The sensitivity is 0.02–0.2 ng/mm² for neutral proteins and ~1.0 ng/mm² for basic proteins.