To calculate the antibody concentration in the antiserum:

  1. Wash the precipitate by resuspending in PBS and re-centrifuging.
  2. Remove the PBS and discard.
  3. Whirlimix each tube to loosen the precipitate.
  4. Add 1ml of 0.1M NaOH to each tube to dissolve the protein precipitate.
  5. Pipette 1ml of 0.1M NaOH into each of 6 cuvettes and 2ml into a seventh to act as a blank. Transfer the contents of each precipitin tube to a corresponding cuvette - ensure that the entire precipitate is dissolved by drawing it up and down in the pipette.
  6. Read the absorption at 280nm (A280) in a spectrophotometer.

Plot a graph of the A280 values against amount of antigen added to each tube

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