Biomedical Engineering Reference
In-Depth Information
References
Aslam M, Dent AH (1998) Bioconjugation. Protein coupling techniques for
the biomedical sciences. Macmillan Reference Ltd., London
Hermanson GT (1996) Bioconjugate techniques. Academic Press, San
Diego
Hosoda H, Ishikawa E (1993) Coupling of haptens to carriers and to
labels. In: Masseyeff RF, Albert WH, Staine NA (eds.) Methods of
immunological analysis. Samples and reagents. VCH, Weinheim, p
465
Hudson L, Hay FC (1990) Practical immunology, 3rd ed. Blackwell, Oxford,
p44
Wong SS (1991) Chemistry of protein conjugation and cross-linking. CRC
Press, Boca Raton
4.1.1 Activation of Proteins with T
RAUT
's Reagent Yielding
Proteins with Additional Free SH Groups
A .9M NaCl, 0.1 M NaHCO
3
,pH8.0
Solutions/Reagents
B .9MNaCl, 10 mMEDTA, disodium salt, 50 mMsodium phos-
phate, 0.02% NaN
3
,pH7.4
C PBS, 10 mM EDTA, disodium salt, 0.02% NaN
3
Mix 4 mg of KLH or a respective amount of a glycerol-containing
solution of KLH (volume maximal 60
µ
µ
l Soln. A. Weigh
0.6 mg Traut's reagent (2-iminothiolane) into an Eppendorf tube
and add the carrier protein solution. Shake at RT for 30 min.
Equilibrate a 10-ml Sephadex G-25 column with Soln. B and
determine the void volume using dextran blue. Apply the reaction
mixture and elute with Soln. B. Use the fractions containing the
activated protein (in the case of KLH a light blue solution) imme-
diately for conjugation (e.g., Protocol 4.1.3).
If BSA or ovalbumin is used, dissolve and elute the protein in
Soln. C and monitor elution by UV reading.
Conjugate MCA-Gly peptides or maleimid-activated proteins
to the iminothiolane-acivated carriers.
l)with190
4.1.2 Conjugation of MCA-Gly Peptides
to SH-Carrying Proteins
We i g h 4 mg of a monochloroacetylglycyl peptide (MCA-Gly pep-
tide, peptide carrying a MCA-glycyl residue at the N-terminus)
into an Eppendorf tube and add the activated KLH or other
iminothiolane-activated carrier protein. Shake vigorously at RT
for 3 h. Dialyze the reaction mixture twice at RT against PBS for 1 h
each. Calculate protein concentration from 235-, 260-, and 280-nm
readings (cf. Protocol 1.1.7).
Filter the conjugate through a 0.22-
µ
m-tip filter and store in
a refrigerator until immunization. For boostering, aliquot the ster-
ile filtrate and freeze at −70
◦
C or mix with glycerol 1:1 and store at
−20
◦
C.
