Biotinylation approach

The biotinylation approach suffers less than the covalent conjugation method from nonspecific protein adsorption. However, the biotinylation method offers limited load capacity of the molecule, which can result in lower recovery of the... 

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... 

The power of the pooled GST fusion protein approach will increase as new biochemical reagents and assays become available. The development of chemical probes for biological processes, termed chemical biology, is a rapidly advancing field. For example, the chemical synthesis of an active site directed probe for identification of members of the serine hydrolase enzyme family has recently been described (Liu et al., 1999). The activity of the probe is based on the potent and irreversible inhibition of serine hydrolases by fluorophosphate (FP) derivatives such as diisopropyl fluorophosphate. The probe consists of a biotinylated long-chain fluorophosphonate, called FP-biotin (Liu et al., 1999). The FP-biotin was tested on crude tissue extracts from various organs of the rat. These experiments showed that the reagent can react with numerous serine hydrolases in crude extracts and can detect enzymes at subnanomolar... 

A few particularly novel approaches have been developed to increase the sensitivity (and specificity) involved in the detection of bacteria by MALDI. The use of avadin-biotin technology to recover biotinylated proteins from... 

Using a similar approach, Clq has been modified with biotin-HPDP and allowed to interact with its specific receptor. Subsequent purification of the Clq receptor was accomplished through cleavage of the disulfide bridge of the biotinylation reagent (Ghebrehiwet et al., 1988). 

Figure 22.21 Antibodies may be conjugated to liposomes using an indirect approach incorporating a (strept)avidin-biotin system. Biotinylated liposomes may be complexed with biotinylated antibodies using (strept)avidin as a bridging molecule or may be complexed with an antibody-(strept)avidin conjugate.

Rothenberg, B.E., Hayes, B.K., Toomre, D., Manzi, A.E., and Varki, A. (1993) Biotinylated diaminopyri-dine An approach to tagging oligosaccharides and exploring their biology. PNAS 90, 11939-11943. 

A very interesting approach was presented recently by Niemeyer et al. [113]. They prepared covalent DNA-streptavidin conjugates to which biotinylated alkaline phosphatase, beta-galactosidase, and horseradish peroxidase, as well as biotinylated anti-mouse and anti-rabbit immunoglobulins, were coupled. Immobilization of DNA-streptavidin conjugates was performed by hybridization with the complementary oligonucleotides, bound to the surface. It was demonstrated... 

The above represent the past and present of the most common enzyme-mediated methods of antigen detection. There are alternate procedures available, involving such methods as antibiotin antibody steps that combine the avidin-biotin systems with a further antibiotin/antienzyme sandwich for still greater sensitivity. Also, there are methods that follow a PAP procedure with a biotinylated antibody to the PAP immunoglobulin followed by ABC detection (15). The obvious problem created with this approach is the tremendous... 

Photo-polymerization was conducted at 254 nm under a modified fluorescent microscope. Allyl-oligonucleotides were employed with methylene blue as the free radical initiator for crosslinking. In the case of proteins, acryloyl streptavidin was first immobilized so that biotinylated proteins could be applied to the gel pad (Vasiliskov et al., 1999). One of the major drawbacks with the gel pad approach was that separate pads had to be manufactured instead of coating the enhre slide with the gel and then printing down the microarray. 

In this approach, the phage antibodies react with biotinylated antigen in solution, and the complex is then captured using Streptavidin coupled to Dynabeads. 

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... 

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