Sulfo-NHS—biotin

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... 

Figure 3. E. chrysanthemi cell surface labelling with sulfo-NHS-biotin. After labelling, the proteins were separated by SDS-PAGE, blotted onto nitrocellulose and revealed with PemB-antibodies (A) or with streptavidin-peroxidasc (B). Lane 1 A350 (wild type) lane 2 A837 kdgRy, lane 3 A350/pPME6. An arrowhead indicates the PemB position.

A water-soluble analog of NHS-biotin containing a negatively charged sulfonate group on its NHS ring structure also is available. Sulfo-NHS-biotin may be added directly to aqueous reactions without the need for organic solvent dissolution. A concentrated stock solution may be prepared in water to facilitate the addition of a small quantity to a reaction, but hydrolysis of the NHS ester will occur at a rapid rate, so the solution must be used immediately. 

The only disadvantage to the use of NHS-biotin or sulfo-NHS-biotin is the lack of a long spacer group off the valeric acid side chain. Since the binding site for biotin on avidin and streptavidin is somewhat below the surface of the proteins, some biotinylated molecules may not interact as efficiently with (strept)avidin as when longer cross-bridges are used (Green et al., 1971 Bonnard et al., 1984). 

The following protocol is a generalized method for the biotinylation of a protein using sulfo-NHS-biotin. 

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. 

With mixing, add a quantity of the sulfo-NHS-biotin solution to the protein solution to obtain a 12- to 20-fold molar excess of biotinylation reagent over the quantity of protein present. For instance, for an immunoglobulin (MW 150,000) at a concentration of 10 mg/ml, 20 pi of a sulfo-NHS-biotin solution (containing 8 X 10-4 mmol) should be added per ml of antibody solution to obtain a 12-fold molar excess. For more dilute protein solutions (i.e., 1-2 mg/ml), increased amounts of biotinylation reagent may be required (i.e., 20-fold molar excess or more) to obtain similar incorporation yields as when using more concentrated protein solutions. 

Instead of BNHS the more hydrophilic derivative sulfo-NHS-biotin (M 443.42) or a spacer derivative, e.g., sulfo-NHS-LC-biotin maybe used. 

Protocol 2. Biotinylation of Target Protein using EZ-Link Sulfo-NHS-Biotin... 

Add the corresponding amount of EZ-Link Sulfo-NHS-Biotin, to get a 10-folcl molar excess over the target protein, directly to the protein solution. Incubate at room temperature for 30 min or on ice for 2h. 

Add 5 0 xl of the NHS-LC-biotin solution in DMF to each milliliter of the protein solution in two aliquots apportioned 10 min apart. Alternatively, add a quantity of the sulfo-NHS-biotin solution prepared in water to the protein solution to obtain a 12- to 20-fold molar excess of biotinylation reagent over the quantity of... 

Activated biotin derivatives e.g., Biotin-ONp (EMD Biosciences) or sulfo-NHS-biotin (Pierce). 

Key words Biotinylation, Antibody, Immunoglobulin, IgG, Biotin, Avidin, Streptavidin, NHS-biotin, sulfo-NHS-biotin... 

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