Biotin-EDTA

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... 

Purify the SH-labeled oligo by gel filtration on a desalting resin using 10 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. The probe now may be used to conjugate with an activated enzyme, biotin, fluorescent tag, or other molecules containing a sulfhydryl-reactive group. 

Quantitative assay of biotinidase activity including evaluation for the presence of a Km defect requires 1 ml of plasma. Serum samples can be also used. Biotin therapy does not affect the assay [ 32 ]. The activity in plasma may decrease during storage (see Pitfalls and Limitations , below). We recommend the use of ethylene-diami-netetraacetic acid (EDTA)-plasma separated from whole blood and assayed within... 

Prepare the antibody for reduction. For labeling IgG antibodies with thiol-reactive reagents, the antibody should be at 5-10 mg of pro-tein/mL in 20 mM sodium or potassium phosphate buffer, pH 7.5-8.0 containing 150 mill NaCl or 10 mM phosphate-buffered saline ethylene diamine tetraacetic acid (PBS-EDTA). When biotin maleimide or biocytin maleimide derivatives are used, the buffer used for the reduction and subsequent steps should be at pH 6.5-7.2. 

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