Biotinylation compounds

FIG. 6 Successive coupling of two different biotinylated compounds with the DNA-STV conjugates 2 [33]. In a first step, a macromolecular functional component (FC, represented by the shaded ellipse), such as a biotinylated enzyme or oligonucleotide, is coupled. In a second step, a biotinylated low-molecular-weight modulator M, represented by the shaded sphere) is coupled to the remaining free biotin-binding sites. The modulator is used to modify the conjugate s hybridization properties or to supplement its functionality. 

Psoralen, or derivatives of 9-methoxy-7H-furo[3,2-g]chromen-7-one tricyclic ring structures, are used as photoreactive groups in crosslinkers, biotinylation compounds, and nucleic acid probes. Psoralens have been used for many years as photochemotherapy agents for treatment of psoriasis and vitiligo (Smith and Barker, 2006). Psoralens react when exposed to UV light... 

Wash the cells several times with PBS, pH 7.4, to remove excess biotinylation compound. 

The nitrogen group in fulleropyrrolidines can be used for conjugation with crosslinking agents or hydrophilic biotinylation compounds for subsequent use in bioconjugation reactions. 

Figure 16.1 The general design of an ICAT reagent consists of a biotinylation compound with a spacer arm containing stable isotope substitutions. The reactive group is used to label proteins or peptides at particular functional groups and the biotin affinity tag is used to isolate labeled molecules using immobilized (strept)avidin.

The use of discrete PEG spacers in the construction of biotinylation compounds not only increases the water solubility of the modification reagent itself, but significantly increases the hydrophilicity and stability of proteins modified with them. Even when high modification levels... 

The NHS ester compounds are sensitive to hydrolysis in aqueous solution, and they likely will hydrolyze faster than more hydrophobic biotinylation compounds due to their hydrophilic-ity. If a stock solution is made at a higher concentration to facilitate the addition of a small amount to a reaction solution, the initial solution should be made in a water-miscible organic solvent that is dried with a molecular sieve. Suitable solvents include DMAC, DMSO, or DMF. If using DMF, use only highly pure solvent, as it may contain amines that can react with the NHS ester groups (Figure 18.13). 

Proteins biotinylated with this reagent will have a characteristic absorbance band at 354 nm, which can be used to determine accurately the number of biotin groups per molecule. No other biotinylation compound has such built-in quantification capability. This feature eliminates the need to consume conjugate by doing a HABA assay to test for the level of biotin incorporation (Chapter 23, Section 7). 

Chapter 11 and Chapter 18, Section 3, describe the major biotinylation compounds and their properties. Also provided in these sections are suggested protocols for reacting each of these reagents with specific functionalities on macromolecules. 

Load the column with the mixture to be purified Elute any unbound protein by adding 20 mM sodium phosphate, 0.5MNaCl, pH 7 5. Add biotin to the same buffer to obtain a final concentration of 0.8 mM to elute the biotinylated compound. 

Another important application of this interaction is in the formation of self-assembled bilayers on silver or gold surfaces. Ebersole et al. [457] showed that avidin and streptavidin molecules will adsorb onto clean layers of Au or Ag from an aqueous solution. Such layers can then be employed to capture biotinylated compounds. These authors used this technique to attach fragments of nucleic acid derived from the herpes virus to a solid support. A more popular approach to this general problem has, however, been to start by adsorption of a suitable biotin... 

Modifications done with biotin-HPDP produce biotinylated compounds with long spacer arms (29.2 A), ensuring good binding efficiency with avidin or streptavidin... 

Furthermore, a simultaneous multiplex detection of several antibodies is impossible with the sequential incubation of biotinylated compounds and STV, as there is no selective linkage between a specific antibody and an individual DNA marker. 

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