1. Prepare the antibody for reduction. For labeling IgG antibodies with thiol-reactive reagents, the antibody should be at 5-10 mg of protein/mL in 20 ml sodium or potassium phosphate buffer, pH 7.58.0 containing 150 ml NaCl or 10 ml 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.
2. Reduce the antibody starting material. Add 2-mercaptoethylamine HCl (2-MEA, cysteamine HCl) to the antibody solution to a final concentration of 6-7 mg/mL (approx 53-62 mM) while stirring. Dithiothreitol (DTT) at a final concentration of 50 mM also can be used for reduction.
3. If 2-MEA is used, incubate the reaction mixture at 37°C for 90 min with stirring or gentle shaking. Incubate the mixture for 30 min at room temperature if DTT is used for reduction.
4. Remove excess 2-MEA (or DTT) from the reduced antibody (desalting). This step is easily performed on a gel filtration column equilibrated with the appropriate PBS (see step 1 ) containing 5 mM EDTA (also see Note 4). The presence of the reduced antibody in the column effluent should be monitored by A280. Because any residual 2-MEA (or DTT) will inhibit the subsequent labeling reaction, it is important to pool only the most concentrated protein fractions eluting earliest from the desalting column. Desalting by dialysis versus the appropriate PBS containing 5 mM EDTA may be performed. In this case, the dialysis buffer should be changed frequently to assure that the 2-MEA (or DTT) has been completely removed.
5. Concentrate the desalted antibody, if necessary, to 10-20 mg/mL (see Note 5). Biotinylate the reduced antibody immediately to avoid reoxidation of the thiols.
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