1. Equilibrate membranes in buffer 6 for 5 min.
2. Block the membranes at room temperature with 3% (w/v) blocking reagent in buffer 6 for 30-45 min.
3. Incubate membranes at room temperature for 30 min with antibody fragments diluted in 3% blocking reagent in buffer 6.
4. Incubate membranes in buffer 6 + 0.3% (w/v) Tween-20, twice for 15-20 min to wash off excess antibody.
5. Equilibrate membranes in buffer 7 for 5 min.
6. Wash the membrane with CSPD diluted in buffer 7 for 5 min in the dark.
7. Blot the membrane to remove any excess liquid before sealing the membrane in a plastic bag and incubating for 15 min at 37°C in the dark.
8. Expose the membrane to X-ray film for between 15 min and 3 h, and develop (see Notes 1-4).
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