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 pL of the solution into a 1.5-mL Eppendorf tube, and evaporate to dryness.
3. Add about 30 pg of antibody in 200 pL PBS to the Iodogen-coated tube with 400 pCi of 125I at room temperature for 10 min.
4. 200 pL 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 pL 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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