Editing Peptide Conjugation to KLH (section)

== Protocol 4 ==

1. Dissolve KLH in 10 mM NaPO4 buffer, pH 7.2 to final conc 16 mg/ml<br/>&nbsp;2. Make 280 mg/ml of MBS in Dimethylformamide freshly<br/>&nbsp;3, Add 10 ul of MBS while stirring to avoid a high local concentration and contine to stire for 30 min at room temperature<br/>&nbsp;4, Centrifuge at 15000 rpm at 4 oC for 5 min. Take a supernatant<br/>&nbsp;5. Separate the MBS-activated KLH from the free MBS by gel filtration on&nbsp;&nbsp;G25 column equilibrated with 50 mM sodium phosphate buffer, pH 6.0<br/>&nbsp;6. Pool MBS-activated KLH and divide equally into 10 microcentrifuge tubes. Store at -80oC until use<br/>&nbsp;7. Thaw MBS-activated KLH in one tube<br/>&nbsp;8. Add 0.5 vol of 0.2 M Na2HPO4 (pH should be 7.3 to 7.5)<br/>&nbsp;9. Dissolve 1 mg of the peptide containing cystein residue at the N- or C-terminus in 0.5 ml of&nbsp;&nbsp;distilled water, and add to MBS-activated KLH<br/>&nbsp;10 Stir the reaction for 3 hours at room temperature<br/>&nbsp;11. Divide equally into 10 microcentrifuge tubes. Store at -80oC until use

=== &nbsp;Deoxidation of Peptide ===

<br/>&nbsp;1. Use Ellman's reagent to determine if there are free sulfhydryls on the terminus of the pep<br/>&nbsp;2. Dissolve 1 mg of the peptide containing cysteine residue at the N- or C-terminus in 0.5 ml of 20 mM NaPO4, pH 8.5<br/>&nbsp;2. Add NaBH4 to a final concentration of 0.1 M<br/>&nbsp;3. Stand the reaction for 45 min at room temperature<br/>&nbsp;4. Adjust the final pH to 4.0 by adding 0.1 N HCl<br/>&nbsp;5. Stand the reaction for 5 min at room temperature<br/>&nbsp;6. Adjust the final pH to 7.5 by adding 0.2 M Na2HPO4<br/>&nbsp;7. Add to MBS-activated KLH<br/>&nbsp;8. Stire the reaction for 3 hour at room termperature<br/>&nbsp;9. Dialyze against PBS at 4 oC overnight<br/>&nbsp;10 Divide equally into 10 tubes, store at -80 Oc until use

=== &nbsp;Immunization ===

&nbsp;2 or more female rabbits (weight 2.5 Kg) are immunized subcutaneously&nbsp;&nbsp;by standard protocol with ~100 ug of peptide-KLH conjugate emulsified with<br/>&nbsp;Freund's complete adjuvant (FCA) 4 times at biweekly intervals, and boosted with&nbsp;&nbsp;peptide-KLH conjugate in Freund's incomplete adjuvant.&nbsp;&nbsp;One week after boost, bleed abou 70 ml from marginal ear vein of rabbits

4 or more female Balb/c mice (4-5 wk) are immunized at the footpad with 10-20 ug&nbsp;&nbsp;of peptide-KLH conjugate emulsified with Freund's complete adjuvant (FCA) twice at&nbsp;&nbsp;4-d intervals. 3 d after last immunization, boosted with ~20 ug of&nbsp;&nbsp;peptide-KLH conjugate in Freund's incomplete adjuvant