Immunoblotting

1. After electophoresis, the proteins within the gel are transferred onto a nitrocellullose membrane using a semidry electroblotter in semidry blotting buffer at 0.3 A for 40 min (see Subheading 2.5. and Note 15).

2. The remaining protein bindings sites on the membrane are blocked for 1 h in Western blocking buffer.

3. Rinse once in PBS and twice in Western washing buffer.

4. Incubate in 0.1-2 ^g/mL of specific antibody in Western washing buffer containing 0.5% (w/v) milk powder for 1 h at room temperature. The sensitivity may be increased by incubating overnight at 4°C. However the background may also increase.

5. Wash three times for 5 min in Western washing buffer.

6. Incubate with the second antibody (e.g., antimouse HRP-labeled secondary antibody for primary MAbs) in Western washing buffer containing 0.5% (w/v) milk powder for 1 h at room temperature.

7. Wash six times for 5 min in Western washing buffer.

8. Develop in ECL reagent for 1 min, and expose to X-ray film (see Note 16 for alternative methods).

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