Photobiotin

N-(4-azido-2-nitrophenyl)-aminopropyl-N -(N-d-biotinyl-3-aminopropyl)-N -methyl-1,3-propanediamine MW 533.65 30.0 A 

Photobiotin can be dissolved in water or buffer at a concentration of 1 mg/ml and stored in the dark at — 20°C until needed. As long as no exposure to light is permitted, the compound is stable for at least 1 year under these conditions. 

The protocol for modifying DNA probes with photobiotin can be found in Chapter 27, Section 2.3. It is based on the method of Forster et al. (1985). The following method is a suggested protocol for the modification of proteins using a photoreactive biotin derivative. Some optimization may be necessary to obtain the best incorporation levels. 

Dissolve the protein to be biotinylated at a concentration of at least 1 mg/ml in water or dilute buffer at neutral pH. 

In subdued light, dissolve photobiotin (Thermo Fisher) in water at a concentration of 1 mg/ml. 

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Figure 11.14 Photobiotin can be made to couple spontaneously with nucleophiles by exposure to UV light. The phenyl azide ring undergoes ring expansion to a highly reactive dehydroazepine intermediate, which can react with amines.

Add a quantity of photobiotin solution to the protein solution to give at least a 5-fold molar excess of biotinylation reagent. 

Remove excess photobiotin by dialysis or gel filtration using a desalting column. 

Psoralen-PEOj-Biotin has been used to label double-stranded DNA for detection using (strept)avidin reagents (Henriksen et al., 1991 Wygrecka et al., 2007). The psoralen photoreactive group provides better insertion yields than typical phenyl azide-based systems, such as the standard photobiotin probe discussed previously in this section. 

When photobiotin is irradiated in the presence of DNA the reaction process nonselectively couples a biotin label to every 100-200 base residues. The result is an oligonucleotide probe detectable by the use of (strept)avidin conjugates. The uses of photobiotin for DNA or RNA modification are summarized in Chapter 11, Section 4. 

To extract excess photobiotin, add 100 pi of 2-butanol. Mix well and centrifuge. Discard the upper phase. Repeat this process two more times. 

Chetrit, P., Gaudin, V., de Courcel, A., and Vedel, F. (1989) A cross-hybridization method for DNA mapping with photobiotin-labeled probes. Anal. Biochem. 178, 273-275. 

Forster, A.C., Mclnnes, J.L., Skingle, D.C., and Symons, R.H. (1985) Non-radioactive hybridization probes prepared by the chemical labeling of DNA and RNA with a novel reagent, photobiotin. Nucleic Acid Res. 13, 745-761. 

Habili, N., Mclnnes, J.K., and Symons, R.H. (1987) Non-radioactive photobiotin-labelled DNA probes for the routine diagnosis of barley yellow dwarf virus. J. Virol. Meth. 16, 225-237. 

Khan, A.M., and Wright, P.J. (1987) Detection of flavivirus RNA in infected cells using photobiotin-labelled hybridization probes./. Virol. Meth. 15, 121-130. 

Lacey, B., and Grant, W.N. (1987) Photobiotin as a sensitive probe for protein labeling. Anal. Biochem. 163, 151-158. 

Nucleic acids can also be biotinylated by nonenzymatic methods with photobiotin, a photoactivatable biotin analog (6), which can be commercially obtained from BRL, Sigma, and other commercial sources I have not compared the suitability of this method of biotin incorporation with that reported here, but expect that the method would be fully acceptable FMC (Rockland, ME) markets an alternate nonradioactive sequence detection kit known as Chemiprobe. The basis of this system is a chemical modification of cytosine residues m the probe DNA. After hybridization, the probe is detected by means of a monoclonal antibody that specifically recognizes the sulfonated DNA. Detection of the bound monoclonal antibody is achieved by means of an alkaline phosphatase-conjugated second antibody. 

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