Biomedical Engineering Reference
In-Depth Information
For affinity chromatography, avidin or streptavidin are coupled
to supports using Protocol 3.6.2. Biotin is covalently bound to pro-
teins or other primary amino groups bearing molecules using its
N-hydroxsuccinimide ester
4
.
Not only biotinylated proteins, but also nucleic acids or peptides
synthesized in the presence of biotin derivatives or carbohydrate-
biotin conjugates, are separated by immobilized avidin and strep-
tavidin, respectively, or are identified by avidin-enzyme or avidin-
gold conjugates.
Mostly the accessibility of an immobilized ligand to its ligate is
increased if the ligand is bound to the matrix via a spacer. If steric
hindrance is suspected, use a lengthened biotin derivative as N-6-
biotinylamidohexanoyl hydroxysuccinimide (Biotin-LC-NHS).
The following protocol is an example for the biotinylation of
immunoglobulins in a molar ratio of 1 mole IgG to 5 moles biotin.
Depending on the kind of protein, ratios up to 1:20 are possible.
Because at a given pH the reactivity and/or accessibility of NH
2
groups of amino acid side chains of a protein are not the same, the
number of conjugated biotin residues may be altered by variation
of the buffer pH between pH 6 and 9 (the higher pH the larger the
degree of conjugation).
A
PBS or saline
Solutions/Reagents
ester
5
B1mg biotinyl-N-hydroxysuccinimide
(BNHS;
58.6 n
µ
µ
Moles/
l,M
r
341.4) in 50
l DMF (ultrapure or distilled), pre-
pare freshly
µ
Supplement 250
l of antibody solution (about 2 mg of antibody
per milliliter of Soln. A) with 1.1
µ
l of Soln. B and shake protected
from light at RT for 1 h. Dialyze the reaction mixture three times
with a 100-fold volume of Soln. A at 4
◦
C for 6 h each.
Add an equal volume of glycerol to the dialyzed sample and
store at −20
◦
C.
Other proteins, such as, for instance, horseradish peroxidase or
alkaline phosphatase, are labeled (conjugated) in the same way.
References
Bayer EA, Wilchek M (1980) In: Glick D (ed.) Methods of biochemical
analysis, vol. 26. Wiley, New York, p 1
Savage MD, Mattson G, Desai S, Nielander GW, Morgensen S, Conklin
EJ (1992) Avidin-biotin chemistry: a handbook. Pierce Chem. Comp.,
Rockford, Illinois, p 273
Hermanson GT (1996) Bioconjugate techniques. Academic Press, San
Diego, p 377
4
The preparation of NHS-biotin is described in: Bayer E, Wilchek M
(1974) Meth Enzymol 34:265
5
Instead of BNHS the more hydrophilic derivative sulfo-NHS-biotin (M
r
443.42) or a spacer derivative, e.g., sulfo-NHS-LC-biotin may be used.
