Protein Protocols Protein Protocols

Glycoprofiling and SPR 887
2. Materials
2.1. Reagents
1. 0.05 M N-Hydroxysuccinimide (NHS).
2. 0.2 M N-ethyl-N'(dimethylaminopropyl)-carbodiimide (EDC).
3. 1 M Ethanolamine hydrochloride, pH 8.5.
4. 0.1 M HCl.
5. Running buffer (HBS): 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid
(HEPES), pH 7.4, 150 mM NaCl, 3.4 mM ethylenediaminetetraacetic acid (EDTA),
0.0005% Surfactant P20.
6. 10 mM Sodium acetate, pH 4.0.
7. 10 mM Sodium acetate, pH 4.5.
8. 10 mM Sodium acetate, pH 5.0.
9. 10 mM Sodium acetate, pH 5.5.
10. Lectin buffer: 10 mM sodium acetate, pH 5.0 with cations 2 mM MgCl 2, MnCl 2,
ZnCl 2, CaCl 2.
11. Lectins used: MAA, SNA, Datura stramonium Agglutinin (DSA), ConA, PNA, aleuria
aurantia Agglutinin (AAA).
2.2. Equipment
BIAcore 1000 apparatus (Biacore AB, Uppsala) is fully automated and controlled
by the manufacturer’s software. During the operation of the equipment the output from
the chip is displayed on VDU as resonance units (RUs) vs time.
3. Method
The procedure used for lectin/SPR can be divided into four steps. The first three are
essential for any SPR experiments and the last one is optional.
1. “Preconcentration” step.
2. Ligand immobilization step.
3. Lectin binding.
4. Enzyme treatment.
3.1. Preconcentration
Before immobilizing a ligand to the chip surface a procedure called “preconcentration”
needs to be performed. The latter step is important for efficient chemical immobilization
of the ligand. It is accomplished by passing the ligand over the chip surface and
utilizing the electrostatic attraction between the negative charges on the surface matrix
(carboxymethyl dextran on a CM chip) and positive charges on the ligand at pH values
below the ligand pI. Preconcentration is performed at 25°C. The ligand is injected at
different pHs and the pH that provides the steepest curve is used (see Note 1). The
detailed sequence of steps is as follows:
1. A continuous flow of HBS buffer is applied at flow rate of 10 mL/min.
2. A 2-min pulse of ligand in 10 mM sodium acetate, pH 4.0, is applied.
3. A 2-min pulse of ligand in 10 mM sodium acetate, pH 4.5, follows.