Note: This method provides a general procedure for use with the majority of Serotec reagents. In some cases specific recommendations are provided on product datasheets, and these methods should always be used in conjunction with product and batch specific information provided with each vial. Please note that a certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques - these are guidelines only and may need to be adjusted for particular applications.

General procedure:

1. Following SDS-PAGE, transfer proteins to blotting membrane according to manufacturer's instructions.

2. Ensure that protein transfer is complete by staining with Amido Black for 5 - 10 minutes.

3. Destain thoroughly in a solution of 10% acetic acid, 10% isopropanol until bands are clearly observed. Rinse thoroughly in water.

4. Place membrane into blocking solution for 30 minutes at room temperature.

5. Place membrane into primary antibody diluted in fresh blocking solution (a concentration of 1-10µg/ml is generally acceptable). Incubate overnight at 4°C or for 2 hours at room temperature. Wash the membrane extensively in PBST (4 x 5 minutes).

6. Add appropriate enzyme conjugated secondary antibody diluted in fresh blocking solution and incubate for 30 minutes at room temperature. Wash the membrane extensively in PBST (4 x 5 minutes).

7. Add appropriate enzyme substrate solution and incubate for time period recommended by manufacturer to visualise protein bands.

Solutions used:

PBST

Sodium chloride 8.0g, Potassium chloride 0.2g, Disodium potassium phosphate 1.15g, Potassium dihydrogen phosphate 0.2g, Tween 20 1.0ml, Distilled water 1litre

Amido Black Stain

Amido Black 10g, Methanol 100ml, Glacial acetic acid 100ml, Distilled water 800ml

Blocking solution

Non-fat dried milk 40g, 1M Tris-HCl, pH8.0 25ml, 5M NaCl, 25ml, Tween 20 1ml, Sodium azide 0.5g,

Distilled water 950ml