Hello Mike,
here is the method we use:
- dissolve 5 mg of antibody in 1 ml of distilled water; add 1 ml of
freshly made 100 mM sodium periodate and stir for 1 hour at room temp.
- dialize overnight against at least 100 volumes of 10 mM sodium acetate
buffer, pH=4.5
- bring the pH to 9.5 by addition of 1M NaOH and add your
amine-containing ligand dissolved in 1 ml of 50 mM phosphate buffer,
pH=9.5. Stir for 2 hours at room temp.
- prepare freshly a solution of sodium borohydride (NaBH4, 10 mg/ml in
distilled water) and add 200 microliters of this solution to the
reaction mix. Stir for 2 hours at room temp. then add again 200
microliters of NaBH4 solution and stir overnight.
- dialyze or desalt.
for additional references you may look at:
- Wilson and Nakane in "Immunofluorescence and related techniques" 1978
(W. Knapp, H. Holubar and G. Wick, eds.), p. 215, Elsevier, Amsterdam
- original method described in Nakane and Kawaoi, J. Histochem
Cytochem., 22, 1084 (1974).
I hope this helps.
-- Gottfried Proess Tel.: + 32 4 366 01 50 Fax : + 32 4 365 16 04 E-Mail: g.proess@eurogentec.be Web-Site : http://www.eurogentec.be <==== visit it !