Love:klh conjugation

=KLH conjugation to peptide (SOP#2)= Written 1/8/07 by Craig Story

The following procedure was used to conjugate 6 peptides with terminal cysteines to KLH.

1. Dissolve 21 mg KLH (Pierce # 77600) in 2.0 ml dH2O (two vials of 21 mg/vial)

2. Dissolve 20.0 mg Sulfo-SMCC (Pierce # 22322) in 4.0 ml dH2O.

3. Slowly, with mixing, add 2.0 ml the SMCC solution to each vial of the KLH.

4. Rock, incubate 1 hour at RmT°.

5. While rocking, equilibrate 4 PD-10 columns with 20 ml PBS.

6. After incubation, add 1.0 ml to bring volume to 5.0 ml for each. Load 2.5 ml KLH/SMCC reaction onto each column. Elute with 3.5 ml Phosphate Buffer and collect what comes off column. (Free Sulfo-SMCC stays on column)

7. Split the KLH into 6 equal portions of ~5mg each

8. Dissolve peptides in DMSO (about 10 µl DMSO per mg). Weigh out approx 1.5-2 mg peptide. After they dissolve in DMSO, add ~200 µl PBS pH 7.4. Some peptides are more soluble than others. Warming tube can encourage solubilization.

Note: 5 µmol scale yields about 5 mg of 1000 Da peptide, 7 mg of 1400 Da peptide Pierce suggests up to 20 mg peptide/20 mg KLH Another lab protocol has 1 mg peptide with 5 mg KLH

9. Add each of the six peptide solutions to tubes with KLH with mixing.

10. Incubate RmT° for 2 hours.

For purposes of immunization, there is no reason to dialyze the peptide conjugates. Aliquots of antigen can be snap-frozen and stored at -80 for long-term storage.

If you run a polyacrylamide gel, you will see that the conjugates largely will not enter the gel, as they are so large and heavily crosslinked.