An alternative to ECL is the use of radiolabeled probes, such as 125I labeled protein A or second antibody raised against the detecting antibody, which are also commercially available (see Note 17).
1. Two holes are made in the bottom of a 1.5-mL Eppendorf tube with a 19-gage needle, and the bottom is plugged with a small piece of nylon wool. The Eppendorf is then filled with 1 mL of swollen G25 Sepharose resin.
2. The "column" is washed with 10 mL of PBS containing 10 mg/mL BSA. The column is finally washed with 2 mL of PBS containing 25 mM KI, which has been freshly prepared. Dissolve 1 mg Iodogen in 1 mL of CHCl3 and pipet 4 ||L of the solution into a 1.5-mL Eppendorf tube, and evaporate to dryness.
3. Add about 30 |g of antibody in 200 |L PBS to the Iodogen-coated tube with 400 |Ci of 125I at room temperature for 10 min.
4. 200 | L of the labeled antibody are then loaded onto the G25 Eppendorf "column" and centrifuged at 170g for 1 min in a Falcon 2063 test tube containing 20 |L of 10 mg/mL BSA. The labeled antibody can be stored at 4°C for up to 3 wk, and is used at 5 x 105 cpm/mL in Western blocking buffer containing 1 mg/mL of unla-beled antibody.
Prior to autoradiography, the gel must be dried onto 3MM paper under vacuum at 60°C for 2 h, after fixing for 30 min. The gel is then exposed to X-ray film with an intensifying screen at -70°C (Notes 18 and 19).
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