Subunit stoichiometry determination by chemical cross-linking

This procedure adapted from Kedei, N. et al. (2001) allows the oligomerization status of a protein to be determined using a chemical cross-linking approach. Chemical cross-linkering reagents create covalent links between adjacent subunits of a multiprotein complex that will therefore migrate as one single complex during denaturing polyacrylamide gel electrophoresis (PAGE). The number of subunits is deduced from the molecular mass of each monomer compared to the molecular mass of the cross-linked homo-multimeric complex. In the present study, we applied this approach to determine the subunit stoichiometry of the Orai proteins that constitute the CRAC channel, expressed in Drosophila S2 cells. Two alternative protocols are described: one on total cell lysates; the other on intact cells. We used the following homobifunctional reagents (Pierce): the lysine-reactive N-hydroxysuccinimide esters bis[sulfosuccinimidyl]suberate (BS3, water-soluble, membrane-impermeant, spacer arm length 11.4 Å) and dithiobissuccinimidylpropionate, the lysine-reactive aryl halide 1,5-difluoro-2,4-dinitrobenzene (DFDNB, water-insoluble, membrane-permeant, spacer arm length 3 Å) and the cysteine-reactive maleimide 1,6-bismaleimidohexane (BMH, water-insoluble, membrane-permeant, spacer arm length 11.4 Å).