Biotin labelled samples

Figure 6.2 The trifunctional reagent sulfo-SBED reacts with amine-containing bait proteins via its NHS ester side chain. Subsequent interaction with a protein sample and exposure to UV light can cause crosslink formation with a second interacting protein. The biotin portion provides purification or labeling capability using avidin or streptavidin reagents. The disulfide bond on the NHS ester arm provides cleavability using disulfide reductants, which effectively transfers the biotin label to an unknown interacting protein.

Figure 6.3 Mts-Atf-Biotin can be used to label bait proteins at available thiol groups using the MTS group, which forms a disulfide linkage after reaction. The modified protein then is allowed to interact with a protein sample and photoactivated with UV light to cause a covalent crosslink with any interacting proteins. Cleavage of the disulfide bond effectively transfers the biotin label to the unknown interacting protein.

HSV in human fibroblasts was detected using biotin-labeled HSV DNA probes, streptavidin-HRP complex, and enhanced CL substrate reagent for HRP [56], The presence of HSV DNA was observed in cells infected with clinical samples known to contain the HSV virus fixed at 48 h postinfection, with a sharp topographical localization and a good preservation of cellular morphology. 

Hybridization can also be performed in solution phase. Since the capture probe is in solution, the kinetics of hybridization is faster than when the capture probe is immobilized. In the solution phase hybridization format, the capture probe is labeled with an affinity label such as biotin that captures the sample target sequence. The labeled probe then binds to the sample target sequence to form the sandwich. After the hybridization is complete, the sandwich is transferred to an affinity support such as avidin or streptavidin, which will capture the sandwich through the biotin-labeled capture probe. Sandwich hybridization performed in solution followed by capture on an affinity support has been referred to as affinity-based hybrid collection (Kl). 

The avidin-biotin procedure has been extensively used in hybridization studies. Since the concentration of all analysed DNAs used is identical, the total concentration of the analysed samples is high. The amplification of the base-mismatch recognition event is necessary to improve sensitivity. The use of oligonucleotide-functionahzed Uposomes or biotin-labelled liposomes as probes for the dendritic amphfication of DNA-sensing processes was characterized by Willner and co-workers [62] and showed better performance using QCM than impedance spectroscopy measurements. 

For quantitation of HIV-1 virus load in serum/plasma samples, testing samples in duplicate is recommended. Samples are considered positive and are quantifiable when duplicate test results are positive. Samples with negative or discordant duplicate results are considered Amp-RT negative. Quantitative detection of Amp-RT PCR amplicons is made by an ELISA-based, nonradio-active oligoprobing system. The system uses streptavidin-coated microtiter wells that capture the biotin-labeled PCR product (DIG-detection ELISA). The protocol for the ELISA is as follows ... 

HIAA. A ready-to-use competitive EIA kit has been developed for measuring chemically derivatized 5-HIAA. In this procedure, dichloromethane is used to convert 5-HIAA to its methyl ester during sample preparation. This methylated derivative competes with biotin-labeled 5-HIAA for a limited number of binding sites of an antibody immobilized on microtiter plates. After incubation, the wells are washed to remove unbound biotin-labeled HIAA and then incubated with antibiotin alkaline phosphatase before a p-nitrophenyl phosphate substrate is added. The amount of biotinylated HIAA bound to the antibody is inversely proportional to the 5-HIAA concentration. This EIA method shows excellent correlation with HPLC, and interassay reproducibility coefficient of variation (CV) is about 7% at a 5-HIAA level of 7.5mg/L. 

In order to detect which sequences on the array (the probes ) have been hybridized by the sequences in the sample (the targets ), the later sequences have to be previously labeled. This is done by incorporating fluorescently labeled nucleotides in the cDNA synthesis step (in two-color arrays) or the incorporation of a biotin-labeled nucleotide in the cRNA synthesis step, as done by Affymetrix (47). 

After dehydrogenation to 234, X = SCoA, the catabolism of leucine 205 (Scheme 62c) differs from that of the other branched-chain amino acids. A biotin-dependent carboxylation leads to the acid 236, X = SCoA, which is hydrated to HMG-CoA 237, a compound involved in isoprenoid biosynthesis. Feeding stereospecifically labeled samples of leucine in studies of terpenoid biosynthesis indicated that the ( )-methyl group was carboxylated without isomerization of the double bond (181, 182). Messner, Cornforth et al. (215) investigated the hydration 236 = 237 catalyzed by the enzyme 3-methyl-glutaconyl-CoA hydratase (EC 4. 2. 1. 18) and showed that the reversible reaction had syn stereospecificity. 

The result is high-quality arrays that can be used with a fluorescence detection system in a manner similar to the glass slide microarrays, although only single color detection is used. cDNA is synthesized from a test RNA sample by reverse transcription followed by second strand synthesis. This is then used as a template for an in vitro transcription reaction to produce biotin-labeled cRNA. As well as introducing the label for detection, this step permits amplification of the RNA and thus enables less starting material to be used. The cRNA is hybridized to the GeneChip and the biotin detected with an avidin-... 

A more recent separation technique uses the so-called isotope coded affinity tags (ICAT Applied Biosystems, Foster City, CA). In this technique, a tag comprising a stable isotopically labeled marker and a biotin molecule is reacted with the protein extract from a perturbed sample whilst an unlabelled tag is used to mark proteins from a control sample. Following purification on an affinity column to isolate the biotin labeled peptides the biotin is cleaved leaving a mixture of tagged peptides. These either have a labeled (clinical sample) or unlabelled marker (control). The mixture is subject to LC-MS(/MS)... 

Before hybridization to a microarray, the biotin-labeled RNA must be purified away from unincorporated nucleotides. It is inadvisable to perform this operation using a phenol chloroform approach because the biotin causes some of the RNA to be partitioned into the organic phase, thus lowering the yield. Instead, use the same cleanup columns used to purify the cDNA reaction (GeneChip sample cleanup module, Affymetrix). Before proceeding, ensure that buffers have been diluted with ethanol where appropriate. This protocol is supplied by the manufacturer and is described here with minor modification... 

A variation to this method to increase its sensitivity has been made through the use of biotin-avidin system. The above sandwich method is followed as it is up to the incubation of test sample reagent with the solid phase (step (11)1. Subsequent a biotin-labeled antibocfy is reacted. Finally an avidin-linked enzyme solution is incubated. 

The corresponding acid promotes the growth of E coli C 124 with an external concentration of 0.5 ng/ml similar to the concentrations used for dethiobiotin or biotin. The generation times are of the same order of magnitude. The conversion of X into biotin by growing cells of E coli C 124 has been established. If a doubly labelled sample of dethiobiotin is incubated =... 

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