Antibodies biotinylated, preparation

Add 50 pi of the NHS-LC-biotin solution in DMF to each ml of the protein solution in two aliquots apportioned 10 minutes apart. Alternatively, add a quantity of the sulfo-NHS-biotin solution prepared in water to the protein solution to obtain a 12- to 20-fold molar excess of biotinylation reagent over the quantity of protein present. For instance, for an immunoglobulin (MW 150,000) at a concentration of 10 mg/ml, 20 pi of the sulfo-NHS-biotin solution (8 X 10-4 mmol) should be added per ml of antibody solution to obtain a 12-fold molar excess. 

The following three sections describe the preparation and properties of fluorescent, radio-labeled, and biotinylated antibodies. 

Prepare biotinylated secondary antibody solution by diluting 2 drops biotinylated antibody with 50 to 100 ml TTBS (nitrocellulose or PVDF) or TBS (nylon). 

Modular construction of the ternary complex begins with the epitope tag immobilization of Gfly subunits on beads. Scheme A represents the display of > d 1, 4) subunits tethered to the bead, by an epitope tag (hexahistidine or FLAG), with soluble oq subunit completing the hetero-trimer assembly (G-beads). The typical G-bead sample preparation involves the initial capture and immobilization of the fly subunits, on beads bearing chelated nickel or biotinylated M2 anti-FLAG antibodies. The bead samples are then washed and resuspended in buffer. For the anti-FLAG... 

Especially useful is the possibility of preparing conjugates of protein/STV chimeras and biotinylated DNA previous to their application in the assay, thus minimizing the amount of incubation steps required for IPCR (see also Section 2.1.4). Therefore, it could be concluded that if a custom-made protein chimera is accessible as a laboratory tool, or if the facilities and experience for preparing such a reagent are available, and additionally, no capture antibodies were needed, these conjugates allow for a very smart and robust approach to ultrasensitive protein detection by IPCR. 

Furthermore, the protocol of the Universal-IPCR simplifies the exchange of the DNA marker if several biotinylated DNAs were prepared. This flexibility in exchange of biotinylated DNA or biotinylated antibody for adaptation of a common protocol to separate applications marks the Universal-IPCR method as an ideal research tool, underlined by the multitude of applications carried out with this method (see Table 1). 

The preparation of biotinylated antibodies is very simple in view of the commercial availability of activated biotin (iV-hydroxysucdnimidobio-tin)41 Biotin conjugates can be stored for several months at —20° without loss of activity. 

This strategy has been applied to select catalytic antibodies from phage-displayed libraries. Two catalytic single-chain antibodies catalyzing the hydrolysis of ampicillin with rate accelerations kcit/kunc lt of 5200 and 320 (kcat = 0.29 and 0.018min-1) were isolated from combinatorial libraries prepared from mice immunized with penam sulfone conjugates and selected with a biotinylated penam sulfone [61]. 

Preparation of Biotinylated Antibodies and Antigens via lysine Residues (see Notes 5 and 6)... 

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