Biotinylation reagent 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. 

Prepare a 50mM solution of hydrazideTTXTpbiotin in DMAC, DMSO, or DMF. Add a quantity of this solution to the purified, oxidized protein to provide at least a 10-fold molar excess of biotinylation reagent over the concentration of protein present. 

The following sections discuss the concept and use of the (strept)avidin-biotin interaction in bioconjugate techniques. Preparation of biotinylated molecules and (strept)avidin conjugates also are reviewed with suggested protocols. For a discussion of the major biotinylation reagents, see Chapter 11 and Chapter 18, Section 3. 

Prepare diazotized p-aminobenzoyl biocytin by using the protocol outlined in Chapter 11, Section 5, but instead of starting with 2mg the biotinylation reagent dissolved in 40 il of IN HC1, use 9mg in 180pi of IN HC1. Proportionally scale up the other reactant quantities used in the protocol. After the reaction is complete, immediately adjust the pH of the final solution to 9. 

Perhaps the most common method of DNA biotinylation is through enzymatic incorporation with the use of a biotin-labeled deoxynucleoside triphosphate. First reported by Langer et al. and Leary et al. in 1981, the procedure is probably the most popular nonradioactive labeling technique reported for oligonucleotide probes. Although biotinylated derivatives of dCTP and dATP are reported in the literature, by far the most frequently employed derivative is biotin—dUTP prepared from the reaction of an amine-modified dUTP with an amine-reactive biotinylation reagent, such as NHS-LC—biotin (Chapter 8, Section 3.1). 

Prepare the biotinylating reagent immediately prior to use. Dissolve Reagent I in DMSO at a concentration of 2 mg/ml or Reagent II in water at a concentration of 2 mg/ml. 

Immediately before use, dissolve sulfo-NHS-biotin (Thermo Fisher) in water at a concentration of 20 mg/ml. Alternatively, the compound may be dissolved in organic solvent to prevent hydrolysis prior to a reaction (i.e., dry DMF or DMSO). Adjust the concentration and quantity of this stock solution to be prepared according to the amount of reagent needed to biotinylate the desired amount of protein. If prepared in water, the sulfo-NHS-biotin stock solution must be used immediately, since the NHS ester is subject to hydrolysis in aqueous environments. 

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. 

Biotin is a common reagent for labelling oligonucleotides, and a new amidite (175) has been prepared for biotinylated oligonucleotides, but the biotin group may be removed by fluoride treatment.Biotinylated ddNTPs have been used for single base extension for multiplex genotyping by mass spectros-... 

An alternative reagent (216) has been prepared for the synthesis of biotinylated oligonucleotides. In this case the protection used for the biotin moiety is base labile rather than the conventional dimethoxytrityl. The photo-crosslinking behaviour of oligonucleotide constructs, incorporating photoactive residues 217a-d at a defined position, has been examined in the presence of their DNA and RNA complementary targets. The X-ray crystal-structure of the photoproduct formed between 4-thiothymidine and adenosine upon near UV irradiation has been reported. 

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