Biomedical Engineering Reference
In-Depth Information
A .5M NaCl in 0.1 M borate, Tricine, TAPS, or carbonate buffer,
Solutions/Reagents
pH 9.5
B .5M NaCl in 0.1 M acetate buffer, pH 4.5
C1M NaCl in pure water
D1M ethanolamine hydrochloride, pH 8.0, in pure water
Wash freshly activated gel (see Protocol 3.6.1) with huge amounts
of ice-cold water or in the case of commercially available activated
gels, e.g., BrCN-activated Sepharose 6B, with ice-cold 0.001 N HCl.
Dissolve the protein (up to 10 mg
/
ml)orthespacer,e.g ,
/
0.2 mg
ml 1,6-diamminohexane, in buffer A. Mix gel and protein
solution in a ratio of 1 g wet gel to 1 ml solution in a flask and incu-
bate at RT for 2 h or at 4
◦
C overnight. Pour the gel on a sintered-
glass funnel and suck off the liquid. Bring the gel back to the flask
and incubate with an equal volume of Soln. D for 2 h at RT (block-
ing remaining active groups). Gently agitate the gel during these
steps.
After the second incubation wash the gel alternating with the
50-fold volume of Soln. A and B, at least twice. Bring the gel to
neutral pH by rinsing with Soln. C.
Store the obtained affinity matrix in a buffer stabilizing the
ligand properties, supplement with a biocide, e.g., 0.02% sodium
azide or 0.1% Thimerosal or some drops of chloroform.
If a spacer such as, for example, 1,6-diaminohexane, was bound
to the matrix, in a second step the carboxyl-groups-bearing ligand
will be immobilized. For ligands with free amino groups the spacer
could be 6-aminohexanoic acid. Coupling reagents often are water-
Water-soluble
carbodiimides
soluble carbodiimides as N-ethyl-N
-(3-dimethylaminopropyl)-
carbodiimide (EDC) or N-cyclohexyl-N
-[2-(4-methylmorpholini-
um)-ethyl]-carbodiimide tosylate (CMC) in slightly acidic buffers,
e.g., 0.25 mm MES or phosphate, pH 4.5.
If the ligand or the coupling reagent is not water soluble, couple
in 80% dimethylformamide (DMF) (v/v) or 80% dimethylsulfoxide
(DMSO) (v/v), adjusted to pH 4.5.
3.6.2.1 Quantitative Determination of Coupled Diamine
Spacers with 2,4,6-Trinitrobenzene Sulfonic Acid
This is a modification of the TNBS procedure by Antoni et al. It
is suitable for polysaccharide media hydrolysable by boiling glacial
acetic acid.
A .5M NaCl in 0.1 M borate buffer, pH 9.5
Solutions/Reagents
2,4,6-trinitrobenzenesulfonic acid
glacial acetic acid
Suspend 100.0 mg wet gel and 15 mg 2,4,6-trinitrobenzensulfonic
acid in 5 ml Soln. A. Centrifuge with 3000
g after 2 h at RT. Wash
the pellet several times with ddH
2
O by suspending and centrifuga-
tion.
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