Immunoblotting

1. Transfer the proteins from the acrylamide gel to nitrocellulose using standard tanktransfer methods.

2. Following transfer, rinse the nitrocellulose with distilled water and stain the membrane with Ponceau S to check for uniform transfer of proteins across the blot. Remove the Ponceau S by rinsing with PBS.

3. Block the membranes in a solution of 5% milk-TBST for at least 1 h at room temperature (see Note 6).

4. Incubate the membrane in primary antibody in a solution of 3% milk-TBST for at least 1 h at room temperature. The following concentrations of primary antibody have been shown to work well for these assays in our laboratory: anti-ptyr (PY20) at 1 ig/mL, anti-ptyr (4G10) at 1 ig/mL, HRP-anti-ptyr (HRP-PY99) at 0.2 ig/mL, anti-IR-b at 1 ig/mL, anti-IRS-1 at 0.5 ig/mL, and anti-IRS-2 at 0.25 ig/mL (see Note 7).

5. Wash the blots three times (10 min/wash) in generous amounts of TBST.

6. Incubate the membranes in secondary antibody in a solution of 3% milk-TBST for 45 min to 2 h at room temperature. The following concentrations of secondary antibody have been shown to work well in our laboratory: HRP-anti-rabbit at 0.1 ig/mL and HRP-anti-mouse at 0.2 ig/mL (see Note 8).

7. Wash the blots three times (10 min/wash) in generous amounts of TBST.

8. Rinse the blots once with PBS.

9. Develop using an enhanced chemiluminescence (ECL) detection kit according to the manufacturer's instructions and expose to film.

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