Biomedical Engineering Reference
In-Depth Information
µ
Dissolve about 20 mg EDAC hydrochloride (M
r
191.7) in 200
l
Universal coupling
protocol
Soln. A immediately before use and mix vigorously with the
peptide-carrier solution. Shake at RT for 2 h.
If necessary, remove surplus reagents and/or make buffer ex-
change by gel filtration on Sephadex G-25 or dialysis. Concentrate
the eluate and the dialysate, respectively, to about 1 ml.
As a variant, activate the peptide separately first and couple then
to the carrier: Dissolve the peptide to 1 mg
/
ml in ddH
2
O, add 10 mg
EDAC hydrochloride per milligram of peptide, and adjust pH to
5.0. Incubate at RT for 5 min andcorrectthepHwithdilutedNaOH
during this period. Then add the same volume of carrier protein
solution. The amount of carrier protein should be in a ratio of
40molesofCOOHgroupspermolpeptide(ovalbumin:M
r
42.7 kD,
31 Asp,48Glu
/
/
Mole). Shake
at RT for 4 h and stop the reaction by addition of 1/10 volume of
1 M sodium acetate buffer, pH 4.2. Free the sample from surplus
reagents by gel filtration or dialysis and concentrate to about 1 ml
by ultrafiltration.
Amount and volume of the conjugate are sufficient for im-
munization of two rabbits (first immunization 200
Mole;BSA:M
r
67.7 kD,54Asp,97Glu
µ
l each, first,
µ
second, and third boost, 100
l each).
References
Hermanson G (1996) Bioconjugate techniques. Academic Press, San Diego.
p 170
4.1.6 Conjugation of Horseradish Peroxidase (Glycoproteins)
by Periodate Oxidation
Glycoproteins, such as horseradish peroxidase, are coupled se-
lectively to other proteins or NH
2
-groups bearing molecules via
oligosaccharide side chain oxidation. The vicinal OH groups of
oligosaccharide residues are oxidized by periodate to aldehyde
groups, which react with amines to form imines (Schiff bases).
/
A . 5M sodium metaperiodate (32 mg
ml ddH
2
O)
Solutions/Reagents
B 0mM sodium acetate buffer, pH 4.5
C .2M sodium carbonate buffer, pH 9.5
D 0mM sodium carbonate buffer, pH 9.5
E4mg
/
ml NaBH
4
or NaBH
3
CN or 50 mM ascorbic acid, freshly
prepared in ddH
2
O
F 0mg
/
ml BSA in PBS
PBS
Dissolve 2 mg purified enzyme (“Reinheitszahl” RZ
=
≈
3; RZ
/
/
A
403
A
274
at 0.5 - 1 mg
ml; for purification see Protocol 3.5.2.4) in
µ
0.5 ml ddH
2
O. Add 25
l Soln. A to the stirred enzyme solution.
