ReadiLink™ KLH Conjugation Kit

1. Prepare protein solution
2. Prepare hapten solution
3. Mix protein with hapten into EDC
4. Incubate the reaction at RT for 2 hrs
5. Purify the conjugate by desalting

The following protocol is a general protocol for a wide variety of haptens. Optimize the protocol accordingly for the conjugation efficiencies upon the size and structure of your hapten. Using a molar excess of hapten over carrier protein ensures efficient conjugation. In general, a reaction with equal mass amounts of hapten and carrier protein will achieve sufficient molar excess.

Add 200 µL of ddH₂O into the vial of mcKLH (Component A) to make a 10 mg/mL solution.

Note: mcKLH solution appears translucent to whitish-blue typically. Do not vortex or heat the solution, which will precipitate the carrier.

Dissolve up to 2 mg hapten in 450 µL Conjugation Buffer (Component B).

Note: Some haptens might have limited solubility, use DMSO (≤30% in the final conjugation solution) to dissolve it first. Higher concentrations of DMSO might irreversibly denature the carrier protein.

Add the 450 µL hapten solution into the 200 µL of mcKLH solution to have KLH-Hapten working solution.

1. Dissolve one vial of EDC (Component C, 10 mg) in 1 mL of ddH₂O and immediately add 50 µL of this solution to the KLH-Hapten working solution, mix gently. Incubate at room temperature for 2 hours. Purify the conjugate by desalting to remove non-reacted crosslinker and protein preservative (e.g., sodium azide).
2. Twist off the bottom closure of the desalting column (Component E), and loosen the cap. Place the column in a collection tube.
3. Centrifuge the column at 1,000g for 2 minutes to remove the storage solution.
4. Remove the cap and slowly add 1 mL of purification buffer to the column. Centrifuge at 1,000g for 2 minutes, remove the buffer. Repeat this step for 3 additional times, discarding the buffer from the collection tube.
5. Place the column to a new collection tube, and gently apply the sample into the center of the compact resin bed.
6. Centrifuge the column at 1,000g for 2 minutes to collect the sample.

7. The KLH-Hapten conjugate can now be used for immunization. If the KLH-Hapten conjugate is to be stored for more than a few days, sterile filter the conjugate, and store at 4 °C or -20 °C.

   Note: If the conjugate is to be used within one week, PBS may be used for purification. If the conjugate will be frozen, use the purification buffer salts (Component D) for purification. If DMSO is used in the conjugation, prepare the purification buffer salts with the same percentage of DMSO used for conjugation. This will minimize the precipitation in the column during desalting. If a precipitate formed during conjugation, centrifuge the precipitated material, collect the supernatant and save the precipitate. Purify the supernatant. Combine the precipitate and the purified conjugate.