preparing the antigen mixture:

• use 200 µg to 1 mg of purified recombinant protein (dissolved in PBS or 8M urea, or with powdered polyacrylamid, when excised from a gel)
• add 30 µg of tripeptide (N-muramyl-alanyl-isoglutamin, Sigma)
• add PBS to a final volume of 300 µl
• add 8 µl of 20% Tween-20
• keep on ice
• add 300 µl of Freund's adjuvans (complete for the 1. injection, incomplete one in the following injections)
• mix thoroughly with a syringe that has no rubber head, use 20G canula, push up and down, store for 10 min on ice, again push up and down. In the end the mixture should be white
• store at 4°C
  
• inject in rabbit

• leave the blood for 1 h at 37°C, oder kürzer
• incubate for more than 1 h on ice (agglutination)
• spin at 3k rpm, 10min
• incubate supernatant (serum) for 30 min at 55°C (inactivation of the complement system)
• store at -20°C

Freund's adjuvans complete
Freund's adjuvans incomplete
Acetylmuranamyl-alanyl-isoglutamin: disolve to 5 mg/ml, use 30 µg per immunisation

Coupling of a peptide to KLH

• dissolve KLH protein in 2 ml of dH2O (yields solution of 10 mg/ml in 1xPBS)
• Dissolve 14 mg of Sulfo-SMCC (crosslinker) in 75 ml DMSO
• Add Sulfo-SMCC to KLH and incubate at 25°C for 30 minutes
• Briefly centrifuge to remove aggregates
• Load supernatant onto a G-25 de-salting column equilibrated with 1xPBS
• Yields approximately 3 mls of activated KLH
• Dissolve 5 mg of cysteine-containing peptide in 0.5 ml 1x PBS
• add 5mg (0.75 ml) acitvated KLH to the peptide
• Incubate for 2.5 hours at 25°C
• Dialyse overnight against 1xPBS (14 kD MWCO)
• Peptide now conjugated and ready for immunisation

injection/bleeding schedule:

0. week:  preimmune serum
0. week:  injection
4. week:  2. injection (1. boost)
   + 10 days:   1. bleed
8. week:  3. injection (2. boost)
   + 10 days: 2. bleed
12. week:  4. injection (3. boost)
   + 10 days:  3. bleed