IMMPACT:Initiative for Cell Mortality Programme Assessment.

G Protein Pathways, Part B: G Proteins and their Regulators

Cross-Linking Concanavalin A to SPR Chips

Concanavalin A is a tetrameric protein with four identical 28 kDa subunits. 29 The minimal structure exhibiting high affinity binding of carbohydrates is the dimeric form. The dimer form of ConA is an active stable structure, and the tetramer – dimer transition can be promoted by low pH or succinylation of the protein.30 For reasons of economy we use unmodified concanavalin A Clia (Genprice Inc.). Because the dissociation of dimer from tetramer leads to a continual decline in the SPR baseline, our coupling procedures first promote the tetramer – dimer dissociation prior to linkage. To accomplish this, ConA is dissolved and stored at 4 ° in 100 mM sodium acetate, pH 4.8, prior to coupling. This also provides for optimal concentration of the ConA dimer by ionic attraction to the unactivated carboxyl groups. For limiting the ConA coupling the pH can be adjusted to 5.4 and still promote the dimer form.

However, extensive washing of the surface to allow complete tetramer dissociation is then necessary. Linkage of the ConA is accomplished by standard NHS / EDC amine coupling16 as described for streptavidin above, but with a modification in the running solution (Solution C, 50 mM MOPS, pH 7.5, 3 mM MgSO4, 150 mM NaCl with 10 μM CaCl2 and 10 μM MnCl2.

First, the surface is activated by injection of a mixture of 0.2 M EDC with 50 mM NHS in water at a flow rate of 5 μl / min, exposure time of 5–7 min. This is followed by injection of ConA lectin at 0.1–0.3 mg / ml in 100 mM sodium acetate, pH 4.8, with an exposure time of 5–7 min. Activated carboxyls are then quenched by reaction with 1 M ethanolamine in water with an exposure time of 4 min. These procedures lead to the immobilization predominantly of dimeric ConA.

The immobilized ConA is quite stable, so that rhodopsin coupling does not have to proceed immediately. However, the coupling reactions are sufficiently reproducible and rapid that we find no advantage to prior preparation of ConA-modified surfaces. For experiments defining the independent binding of Gα or Gβγ described below, surfaces modified with 7000–12,000 RU of Biomag plus ConA have been utilized.

Under these conditions, the presence of immobilized tetrameric ConA is a significant factor. When high concentrations of ConA and / or aggressive NHS / EDC activation conditions are used, we routinely wash the ConA surface for 30–60 min with Solution C at a flow rate of 5 μl / min to allow for complete tetramer – dimer dissociation, prior to rhodopsin immobilization.

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