Subject biotinylation

Biotin can be synthesized by the human colon flora. The question to which extent this production contributes to covering the host-organism s requirements is, however, subject to discussion. In most foods of animal origin as well as in cereals, biotin prevails in the protein (= enzyme)-bound form as e-N-biotinyl-L-lysine (= biocytin). Brewer s yeast, liver, soya beans, and peanuts number among the biotin rich foods [1]. 

To check if PemB is surface exposed, E. chrysanthemi cells were subjected to proteolysis. Treatment of the cell suspension with trypsin, proteinase K or chimotrypsin at a concentration of 0.1 to 1 mg/ml for 1 h did not cause PemB proteolysis or its liberation into the medium. Cell pre-treatment with EDTA-lysozyme, which renders the periplasmic proteins accessible to proteases, gave no effect. PemB was also resistant to proteolytic digestion in extract of cells disrupted by sonication or in a French press. Only addition of Triton X-100 (up to 0.1%) causing formation of the micelles with PemB lead to a quick proteolyis of this protein (data not shown). In another approach to analyse the PemB exposition, bacterial cells were labelled with sulfo-NHS-biotin. This compound is unable to cross membranes and biotinylation... 

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. 

This method, either standard or elite (increased molar ratio), remains the forefront of much of the immunocytochemistry being performed today, and will be the main subject of this chapter. There are new methods, though, that are being used with increased frequency, such as the labeled-avidin binding method, sometimes called the streptavidin-binding method, and a newer catalyzed amplification method that uses avidin, biotin, peroxidase, and a biotinyl tyramide to achieve even more sensitivity. These methods will be discussed at the end of this chapter. 

Fig. 12 a Quenching assay where QSY-7-labeled DNA strand (DNA-QTL) is presented to microsphere and microsphere-bound biotinylated ALF capture strand, b Competition assay for detection of target DNA. Variable amounts of ALF target are added to microsphere and bound ALF capture strand. This is then subjected to a fixed amount of QSY-7-labeled DNA. The amount of quenching is dependent on the amount of ALF target already bound on the microsphere. (Reprinted with permission from Ref. [33]. Copyright 2002 American Chemical Society)... 

Fig. 1. Specific identification off. coli containing plasmid pBR322. Approximately 225 colonics, consisting of a 10 1 mixture of plasmid-frec and plasmid-containing cells, was grown on a nitrocellulose filter. The filterwas subjected to the lysis protocol described here, followed by a hybridization with biotinylated pBR322. Sites of positive hybridization were detected by means of streptavidin and alkaline phosphatase. The dark sites correspond to colonies harboring pBR322. Plasmid-free cells give the faint signals present at numerous sites on the filter.

If the ligand protein is fluorescently labeled, resuspend the cells in 10 pL PBS and subject directly to FACS. If the ligand protein is biotinylated (protocol 2), resuspend the cells in 10 pL PBS containing streptavidin, R-phycoerythrin conjugate (1 10 dilution in PBS). Incubate on ice again for 10 min. 

The probes of this type were shown to selectively label at least 75% of human kinases in crude cell lysates, thus demonstrating their selectivity and promiscuity for kinases [101]. As a follow up, the labeled kinases were subjected to proteolytic digestion, and the biotinylated peptides purified on avidin beads and analyzed by LC-MS/MS. This analysis demonstrated that the site of probe labeling was indeed the conserved active-site lysine as predicted. In contrast to the promiscuity demonstrated by the acyl phosphate probes, several selective covalent inhibitors of protein kinases have been used as ABPP probes. Wortmannin is a natural product derived from the fungus Penicillium funiculosum. It is a potent and specific covalent inhibitor of phosphoinositide 3-kinase (PI3K) and the PI3K-related kinase (PIKK) families [102, 103]. The use of natural products in relation to ABPP is covered by Breinbauer et al. [104]. 

Comeal organ culture combined with objectively quantifiable assays for comeal epithelial barrier disruption reduces the high variability associated to the subjectively scored Draize Test. Although the surface biotinylation allows for an objective outcome measure, the scoring system is not yet quantitatively comparable for assessment of ocular irritancy to multiple test products. As it is utilized more extensively in varied laboratories with numerous test chemicals a standardized scoring system can be elicited similar to the familiar Draize Test. 

Nucleic acids have been linked to a variety of surfaces including polystyrene beads, glass, silicon, gold, and even cells. Immobilization of nucleic acids may occur through a number of covalent linkages that are the subject of other chapters. Strept(avidin) may then be bound to a surface through a biotinylated nucleic acid linker. Alternatively, strept(avidin) may be linked to a surface directly with methods used for other proteins. This chapter will describe the biotin-strept(avidin) system and focus on the use of the biotin-strept(avidin) to link nucleic acids to surfaces. 

Figure 11.10. Identification of proteins subject to S-nitrosyla-tion due to nNOS activity, a Experimental strategy. Protein samples were selectively biotinylated as before (cf Figure 11.9), enriched by adsorption to solid phase-bound streptavidin, blotted, and detected by specific antibodies, b Results for several proteins. SM Starting material (not passed through colunm both S-nitrosylated and unmodified protein molecules will show up here). El Colunm eluate - this will only detect the nitrosylated proteins, -i- Samples from wild-type mice (nNOS -I-/-I-), - samples from knockout mice (nNOS -/-). Data reproduced with permission from Nat Cell Biol. 3 193-7 (2001)...

If the identity of the OP-labeled protein is unknown, a tagged OP, for example a biotinylated OP, can be used to identify the protein (Schopfer et ah, 2005). After the identity of the OP-labeled protein is known, identification of the OP-labeled peptide depends on separating it from contaminating peptides. We have found that the OP-labeled peptide is frequently not found by mass spectrometry unless it has been extensively purified. In some cases it is possible to identify the labeled peptide simply by LC-MS-MS, where an enzymatic digest of the isolated protein is subjected to liquid chromatography on a Cl8 nanocolumn and the effluent from the column is electrosprayed directly into the mass spectrometer. For other cases, extensive HPLC puri-flcation of the enzymatic digest is necessary to obtain a purifled fraction of peptides that can be introduced into the mass spectrometer. 

The use of the avidin-biotin system enables a separation of the primary and secondary steps. Previous employment of an antibody-marker for direct detection was problematic since such conjugates are very large and subject to a variety of irrelevant interactions that interfere with the desired immunochemical binding. In contrast, the biotinylated antibody can first be incubated with the cell or tissue sample following this initial interaction, the sample can be fixed and incubated subsequendy with... 

A number of assays for the hydrolase-type of Hyals were developed in the last decade that facilitated their characterization. These include microtiter-based ELISA-like assays, in which a highly specific HA-binding protein substitutes for the antibody component [186]. Biotinylated HA bound to microtiter plates is subjected to Hyal activity, and the remaining HA quantified by an avidin-enzyme color reaction [107]. HA substrate gel zymography procedures were also formulated that facilitated additional studies of these enzymes [187]. 

The amplification reaction is carried out in IX PCR buffer containing 100 yM dNTPs, 30 pmol of biotinylated primers, 10-100 ng of human template DNA, and 2 U of Taq polymerase. The PCR profile consists of an initial denaturation at 95°C for 5 min. The samples are then subjected to PCR with 30-s incubations at 94, 55, and 72 C for 35 cycles. See Note 3 for an alternative method of biotinylating PCR products. A lengthy discussion of optimization of PCR reactions is beyond the scope of this chapter, but further information can be found in Note 5 and references given therein. 

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