Paramagnetic beads coating with streptavidin

Figure 12.5 illustrates schematically the triplex-mediated capture procedure for the purification of specific ds DNA as described by Ito et al. (1992). A biotinylated pyrimidine oligomer is incubated under acidic conditions (pH 4.5-5.0), since the cytosines should be protonated, with a DNA mixture containing the target. The triplex is then captured with paramagnetic beads coated with streptavidin, the beads washed several times and the retained duplex eluted. 

Tris(hydroxymethyl)methylamine (TRIS), sodium chloride, sodium citrate, ethylenediamine tetraacetic acid disodium salt (EDTA), lithium chloride, Tween 20, streptavidin 10 nm colloidal gold labelled, hydrochloric acid (37%), nitric acid, streptavidin-coated paramagnetic beads (MB) with a diameter of 2.8 pm, Dynabeads M-280 Streptavidin (Dynal Biotech, Oslo, Norway) biotinylated probe oligonucleotides which sequences are shown in Table 53.1. 

Fig. 9. Enzyme electrochemiluminescence (ECL) immunoassay protocol, (a) Trinitrotoluene (TNT) and dini-trophenyl of haptenylated dextran compete for antibody-binding sites, (b) Antibodies attached to dextran bound to streptavidin-coated paramagnetic beads, (c) ECL detection of enzyme-labelled antibodies magnetically concentrated on electrode. Reprinted from Wilson et al. [68], Copyright (2003), with permission...

Fig. 3. Functionalization of monomaleimido-Nanogold 1.4nm diameter (A). Schematic representation (not in scale) of the analytical protocol (B) (I) Immobilization of the biotinylated CF-A probe onto streptavidin-coated paramagnetic beads (MB) (II) addition of the Target CF to the first hybridization event (III) addition of monomaleimido-nanogold (AuNPs) functionalized with signalling thiolated CF-B probe to the second hybridization event (IV) accumulation of final conjugate on the surface of the M-GECE and (V) magnetically trigged direct DPV electrochemical detection of AuNPs tags in the conjugate.

A TS-100 ThermoShaker (Spain) is used for all the incubations for the binding of streptavidin coated paramagnetic beads with biotinylated primary antibody. 

Preparation of template Typically for a reaction 15-/tl streptavidin-coated paramagnetic beads (Dynal) are washed twice with 2(X) /il each of the B/W buffer (Dynal), suspended in 15 tl B/W buffer and added to the PCR reaction mixture. After incubation for 30 minutes at room temperature, the supernatant was removed through magnetic separation and the beads incubated with 50 pA 100 mM aqueous NaOH for 5 minutes at room temperature to denature the non-biotinylated DNA... 

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