- Dissolve or dialyze recombinant Anthrax Protective Antigen (rPA) (BEI Resources, #NR-3780) in 0.1 M Carbonate Buffer (pH = 8.8-9.5) to final concentration of 5-10 mg/ml - The pH and final protein conc. will depend on protein used.
- Dissolve NP-OSu (Biosearch Tech, #N-1010-100) or NIP-OSu (Biosearch Tech, #N-1080-100) in N’N’-Dimethylformamide. Final conc. = 10 mg/ml [Higher (i.e. 25 mg/ml) final conc. of hapten solution will help to make higher substitution ratio haptenated protein.]
- Place protein in small reaction vial (wrapped in foil) with constant stirring, and then add hapten solution in dropwise fashion (amount determined for given substitution ratio from above calculations).
- Allow to react at room temperature for 4 h and then transfer reaction mixture to 4°C and incubate overnight with constant stirring.
- Dialyze extensively (3-4 changes) against PBS pH 7.4 at 4°C.
- Calculate substitution ratio by measuring Moles Hapten/Moles carrier.
rPA conc. = A280 = mg/ml rPA Mol.Wt. = 83,000
BSA conc. = A280 = mg/ml BSA Mol.Wt. = 68,000
NP conc. = A430/14.5 = mg/ml NP-OSu Mol.Wt. = 292
NIP conc. = A430/10 = mg/ml NIP-OSu Mol.Wt.= 420