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Sample Protocol: One-Color Detection

  1. Perform electrophoresis.
  2. Remove the stacking gel by cutting it away.

    Note: The stacking gel will exhibit high background when the gel is imaged.

  3. Fix the proteins in the gel by incubating in 50% isopropanol + 5% acetic acid for 15 min with gentle shaking. Then wash with water for 15 min.

    Note: Use clean gloves and incubation trays to keep background low.

  4. Incubate with primary antibody at 4 °C (1 hour to overnight; must be determined experimentally) with gentle shaking.
    • Blocking is not necessary prior to adding primary antibody.
    • Since In-Gel detection is not as sensitive as a standard Western blot, a higher than usual concentration of primary antibody may be needed.
  5. Wash the gel 3 times for 10 min in PBS + 0.1% Tween® 20 (PBS-T) with gentle shaking.
  6. Incubate with secondary antibody for 1 hour with gentle shaking.
  7. Wash the gel 3 times for 10 min in PBS-T with gentle shaking. Then wash in PBS for 5 min.
  8. Image the gel with an Odyssey® Infrared Imaging System.

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