Biomedical Engineering Reference
In-Depth Information
β
4.1.4
-Galactosidase-Immunoglobulin Conjugate
(Coupling via SH Groups)
β
-Galactosidase from
Escherichia coli
contains sufficient SH groups
for conjugation; therefore, only the antibody has to be activated by
introducing maleimide groups which react with the SH groups
of the enzyme. Conjugation of other proteins or haptens may be
performed analogously.
A .1M sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2
Solutions/Reagents
B .1M sodium phosphate, 0.15 M NaCl, pH 7.2
C
storage buffer tenfold
:0.1M sodium phosphate, 1 M NaCl,
10 mM magnesium acetate, 1% NaN
3
(w/v), 10 mg
/
ml BSA,
pH 6.5
/
Dissolve the enzyme to a concentration of 1 - 2 mg
ml in Soln. A (if
it is delivered in another buffer, dialyze against Soln. A). Mix 1 mg
β
-galactosidase in Soln. A with 0.25 mg SMCC activated antibody
(concentration about 1 mg
/
ml; for activation see Protocol 4.1.3).
Shake at RT for 2 h, dialyze or desalt on a Sephadex G-25 column
against Soln. B and concentrate to about 2 mg
/
ml.Mix9vol.of
the concentrated conjugate with 1 vol. Soln. C and store without
further purification at 4
◦
C.
β
4.1.4.1 Enzyme Reaction of
-Galactosidase
A
substrate buffer
:3mM p-nitrophenyl-d-galactoside
(M
r
Solutions/Reagents
β
301.3), 10 mMmagnesium acetate, 10 mM
-mercaptoethanol
ρ
/
(M
r
78,13;
1.114 g
ml)inTBS,pH7.5
B
stop solution
:0.1M EDTA in 2 N NaOH
TBS
µ
Inawellofamicrotiterplateincubate100
l of an appropriate
β
dilution of
-galactosidase conjugate at RT for 30 min.Washthor-
oughly with TBS and start enzyme reaction by addition of 100
µ
l
Soln. A per well. Stop after 5-20 min (the time period has to be the
same for all wells) by addition of 100
µ
l Soln. B per well. Mix and
read O.D. at 405 nm.
4.1.5 Carbodiimide Coupling of Peptides to Carrier Proteins
with 1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide
(EDAC, EDC)
Solutions/Reagents
A .1M MES (4-morpholinoethanesulfonic acid) pH 4.7
Dissolve 4 mg of carrier protein (KLH, ovalbumin, BSA or the like)
in about 400
µ
µ
l of Soln. A
3
. Add a solution of 4 mg peptide in 400
l
Soln.Aandmixwell.Themolarratioshouldbeatleast10moles
of peptide per mole carrier protein.
3
If KLH is used, the buffer should contain 0.9 M NaCl.
