After the antibody starting material has been prepared in the amine-free reaction buffer of choice (see Note 3), the researcher should then calculate the amount of amine-reactive biotin needed.
1. Select the molar ratio of labeling reagent to antibody (MR). The MR represents the number of moles of amine-reactive biotin that are added to each mole of antibody in the reaction mixture. The recommended MR to use depends on the antibody concentration. For IgG antibodies at <1 mg/mL, we recommend a MR of 30-40. Antibodies at 1-3 mg/mL and 4-10 mg/mL should be labeled at a MR of 20-25 and 10-20, respectively.
2. Calculate how much labeling reagent is required to achieve the selected MR. It is convenient to calculate this value based on the quantity of antibody to be labeled:
mg of labeling reagent per mg antibody = (MW of label x MR)/MW of antibody or fragment (Eq. 1)
3. Calculate the total amount of labeling reagent required:
total mg of labeling reagent = answer from Eq. 1 x total mg of antibody (Eq. 2)
4. Calculate the ^L of labeling reagent stock solution to add based on a 10 mg/mL stock solution:
volume of labeling reagent = answer from Eq. 2 x 100 (Eq. 3)
5. Prepare the reactive biotin stock solution. Weigh out slightly more labeling reagent than is actually needed. Dissolve it in the appropriate volume of anhydrous dimethyl sulfoxide (DMSO) or dimethyl-formamide (DMF) to obtain a concentration of 10 mg/mL. Vortex mixing or brief sonication can be used to facilitate dissolution, if necessary. This solution may deteriorate on storage (see Note 6). Note that Eqs. 1-3 can be used to calculate the appropriate amounts to use of all of the other labeling reagents discussed in this chapter. Additional variations in these procedures are discussed in Note 7. Molecular weights of the commonly used reactive biotin derivatives are listed in Table 3.
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