Biomedical Engineering Reference
In-Depth Information
Important
: Prepare conjugates with different carrier proteins for
immunization and testing and use different coupling reagents, if
possible.
References
Lindner W, Robey FA (1987) Int J Peptid Res 30:794
4.1.3 Conjugation of Sulfhydryl Peptides Using
4-(N-Maleimidomethyl)-Cyclohexane-1-Carbonic Acid
N-Hydroxysuccinimide Ester (SMCC)
A 0mM HEPES, pH 7.4
1
Solutions/Reagents
B 0mM HEPES, 10 mM EDTA, pH 6.8
C .1M sodium phosphate, 0.15 M NaCl, pH 7.2
µ
Dissolve 4 mg ofthecarrierproteinin400
l Soln. A. Dissolve about
0.5 mg SMCC (succinimidyl 4-(N-maleimidomethyl)-cyclohexane-
1-carboxylate, M
r
334.3)
2
µ
l DMF and add this solution to
the carrier protein. Shake at RT for 1 h, centrifuge and desalt on
a Sephadex G-25 column, equilibrated with Soln. B (the activated
carrier appears in the void volume). Cool the receiving tube in an
ice bath.
Dissolve 4 mg of a Cys-containing peptide in 400
in 50
µ
l Soln. B and
mix with the solution of the activated carrier protein. Shake at RT
for 1 h, aliquot and freeze at −70
◦
C.
Dialyze an antibody solution against Soln. C and concentrate
Conjugation of
antibodies
/
to 20 - 30 mg
ml.Add3mg of sulfo-SMCC (3-sulfosuccinimidyl-4-
(N-maleimidomethyl)-cyclohexane-1-caboxylate, M
r
436.7) or of
sulfo-GMBS (N-(
γ
-maleimidobutyryloxy)-3-sulfo-N-hydroxysuc-
cinimide ester, M
r
382.3), dissolved in ddH
2
Oto60mg
/
ml,to
10 mg of antibody, rock at RT for 15 min and add further 3 mg
of coupling reagent. Desalt on a Sephadex G-25 column, equili-
brated with Soln. A, after total incubation time of 30 min and use
the activated antibody (protein) for conjugation immediately (see
above).
References
Hermanson G (1996) Bioconjugate techniques. Academic Press, San Diego.
p 235, p 444
1
If KLH is used, the buffer should contain 0.9 M NaCl and 0.25 mg
SMCC/4 mg KLH are used.
2
Instead of the DMF solution of the water-insoluble SMCC, the same
amount of sulfo-SMCC in water my be used.
