Briefly, nuclear extracts containing equal amounts of protein for each sample were incubated with poly (dI-dC) (1 μg/μl) for 5 min, followed by the addition of binding buffer (20 mM HEPES pH 7.9, 1 mM DTT, 0.1 mM EDTA, 50 mM KCl, 5% glycerol and 200 μg/ml BSA) and biotinylated oligo (10 ng/μl).
When triplex formation was followed by addition of nuclear extract, protein binding was indeed inhibited.
Furthermore, previous reports show that suppression in vitro by murine Treg is prevented by addition of IL-2Rγ-chain binding cytokines [17].
DNase I digestion was carried out by addition of the enzyme in binding buffer containing 5 mM CaCl2 followed by incubation for 5 min at 37°C; the reactions were stopped by the addition of 25 mM EDTA.
Similar to mice, we show that naturally occurring human Treg very efficiently suppress both proliferation and cytokine production by effector T cells, which can be reversed by addition of IL-2Rγ-chain binding cytokines.
Radiolabeled RNAs were annealed by heating to 95 °C, snap cooling to 4 °C, addition of binding buffer, followed by equilibration to rt over at least 20 min. Comparison with a slow-cooling annealing procedure produced no difference in protein affinity.
By careful oligonucleotide design, and/or by addition of single-stranded DNA-binding protein, problems with unspecific sequence signals due to mispriming can be reduced.
Formation of pure and mixed gels is markedly disrupted by addition of acetate anions, consistent with binding of acetate to the urea functionalities.
In a control experiment, the specific IgG binding to these cells was calculated by addition of cold human IgG, which inhibited the binding of radioactive IgG to 93% (data not shown).
Such SLBs were then incubated with VLPs, and the site of binding was visualized by addition of rhodamine-labeled phospholipid vesicles containing H type 1 used as labels for the unstained VLPs (red in Figure 4).
