GLUTERALDEHYDE CONJUGATION OF OLIGOPEPTIDES TO PROTEINS FOR ELISA

  1. Dissolve 30 mg of the carrier protein (ex. KLH) in 3 ml of phosphate buffered saline (PBS) The amount of protein should be about 4 times more than you expect to use. Dialyze for 24 hours in PBS. Centrifuge at about 12,000 xg (use 10,000 rpm in an SS-34 rotor) for 20 minutes to remove insoluble material.
  2. Measure the absorbance of the protein solution at A280 after calibrating a spectrophotometer with PBS. For KLH, O.D . = 2.02 for a 1 mg/ml solution.
  3. In the following experiments, we were conjugating a 15 amino acid peptide to KLH. The mass ratio of peptide to carrier protein was 1:2. Conjugation was performed in a 3 ml volume. Add PBS to the KLH, add the peptide to the carrier protein, and then add the gluteraldehyde as a 1:100 dilution in PBS while stirring. Two examples are given below.
    < /TR>
    Example KLH PBS Peptide Gluteraldehyde 1 M Lysine (in H2O)
    1 20 mg 2 ml 10 mg 1 ml 120µl
    2 3.38 mg in 1.2 ml 0.7 ml 1.69 mg in 0.1 ml 1 ml 120µl
  4. Stir at room temperature for 1-6 hours. Check A405 about every 15 minutes to check for a plateau. Add lysine when the OD plateaus to stop the reaction. Continue to stir for another hour.
  5. Dialyze in PBS overnight at 4°C.
  6. Make dilutions of peptide to 100µg/ml, 50µg/ml, 10µg/ml, and 1µg/ml in carbonate coating buffer. For the second conjugation above, 1.69mg/3.12ml = 542 µg/ml. Coat ELISA plate with 50µl/well for 2 hours at room temperature.

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Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Itoh, K., McCubbin, V., and Frank, M. B. Gluteraldehyde Conjugation of Oligopeptides to Proteins for ELISA. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/glutfix.html). 1997. Oklahoma City. Revision Date: October 2, 1997."