Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Protein Staining on Nitrocellulose Paper
Detection Using Silver and Antibody Complexes

Protein bands from gels are transferred onto nitrocellulose paper and stained with a silver complex. Under these conditions, antigenic determinants are preserved, and individual Proteins can be detected via reaction with specific Antibodies using blotting [416].

Electrotransfer of proteins to nitrocellulose. Following Separation by Laemmli SDS-PAGE [207], The transfer of proteins to nitrocellulose paper is carried out as follows. The nitrocellulose paper is soaked in Electrophoresis buffer (24 mM Tris + 192 mM Glycine + 20% mercaptoethanol, pH 8.3) and then placed onto the gel. The resulting gel–nitrocellulose "sandwich" between two layers of Whatman 3MM filter paper (also pre-soaked) is assembled into an electroblotting unit (E-C Apparatus) such that the gel faces the cathode and the nitrocellulose paper faces the anode. The transfer is conducted for 4 h at 5.0 W with continuous buffer Circulation.

Silver staining [281, 253].

1. Wash the protein-bound nitrocellulose paper overnight in 0.01 M Tris-HCl buffer, pH 7.4.

2. Wash twice for 5 min with distilled Water.

3. Incubate in 100 mL of potassium ferro(II) cyanide (0.5%, w/v) for 5 min with gentle agitation.

4. Rinse with distilled water.

5. Transfer to 100 mL of a solution containing 0.2 g silver nitrate, 0.2 g ammonium nitrate, and 0.5 mL of 37% formaldehyde. Agitate the paper in this solution for 20 min.

6. Decant the nitrate solution. Wash the paper twice with distilled water (10 s each) and then once more for 3 min with freshly distilled water.

7. Leave overnight in 100 mL of an aqueous solution containing 3 g sodium carbonate and 0.25 mL of 37% formaldehyde.

8. Wash twice (15 min in total) with distilled water.

9. Place for 15 min in 100 mL of a solution containing 1.4 mL concentrated ammonia, 0.07 g NaOH, and 0.2 g silver nitrate.

10. Rinse twice with distilled water.

11. Place in 100 mL of a solution containing 5.0 mg citric acid and 0.019% formaldehyde. Proteins appear as light bands against a yellowish-brown Background. If the original gel did not contain SDS, the bands stain yellowish-brown and are difficult to distinguish from the background.

Detection using specific antibodies.

Buffer A. 10 mM Tris-HCl (pH 7.5) containing 0.05 M NaCl, 2 mM EDTA, 4% BSA (Fraction V), and 0.1% sodium azide.

Buffer B. 10 mM Tris-HCl (pH 7.5) containing 0.05 M NaCl and 2 mM EDTA.

1. Equilibrate the silver-stained nitrocellulose strip overnight in 0.01 M Tris-HCl (pH 7.4).

2. Transfer to Buffer A and incubate for 24 h with shaking.

3. Incubate for 16 h with shaking in fresh Buffer A containing the appropriate amount of antibodies.

4. Wash with Buffer B (5 times for 10 min each).

5. Incubate in fresh Buffer A containing ¹²⁵I-labeled protein A (Sigma) (2∙10⁵ cpm) for 3–4 h.

6. Wash with Buffer B (6 times for 10 min each).

7. Air-dry.

8. Expose to Kodak film (X-Omat AR) using an intensifying screen at —70°C.