Probing the Blot with Antibodies
Note: standard procedure is a 1 hour pre-block, a 1 hour primary antibody incubation, 4 x 5 minute washes, a 1 hour secondary antibody incubation, 4x 5 minute washes, and development with HRP

  1. Transfer your gel to nitrocellulose or other blotting material. See previous protocols.
  2. Make 100 mL blocking solution of 5% nonfat dry milk in PBST (5g in 100 mL).
  3. Add the blot to ~20mL blocking agent to a boat. Rock for 1 hour at room temperature. Store the excess blocking solution at 4C.
  4. Add the appropriate amount of primary antibody to 2 mL blocking solution.
  5. Cut a sheet protector to fit the blot plus ~1cm excess on each side.
  6. Remove the blot from the pre-block solution. Place it in sheet protector and seal on three (2 long and 1 short) sides. Add ~2 mL antibody + blocking solution to the blot. Remove the large bubbles.
  7. Seal the sheet protector sandwich on the fourth side. Allow to rock at 4C for 1 hour. Keep the rubber bands from impinging upon the blot.
  8. Cut open sheet protector sandwich and pour the primary antibody solution into a beaker on your bench. After removing it from the sheet protector sandwich, place the blot in 20 mL PBST in a boat.
  9. Rock for 5 minutes at room temperature. Remove PBST. Repeat to total of 4 5-minute washes.
  10. Add the appropriate amount of secondary antibody to 20 mL blocking solution.
  11. Add the blot to 20 mL blocking solution in a boat. Rock for 1 hour at room temperature.
  12. Wash in PBST 4 times for 5 minutes each at room temperature.
  13. (Where is this drying step?) Blot the nitrocellulose dry by brushing backside along a Kimwipe and collecting moisture from the edge.
  14. For each blot, immediately before use mix 2.5 mL enhanced luminol reagent (dark bottle, lab stock stored at 4C) and 2.5 mL oxidizing agent (white bottle, lab stock stored at 4C). Incubate the blot in the mixture for 2 minutes.
  15. Image using film or using Alpha Innotech.

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