5LO can bind to nanodiscs in the presence of Ca2+.
(A) Blue native PAGE (4–16% Bis Tris gel) analysis of ND-5LO complexes formed after incubations of ND and 5LO with and without 1 mM Ca2+ present. Lane 1, purified ND. Lane 2, purified 5LO. Lane 3, sample from incubation of 5LO, ND and Ca2+. Each lane is loaded with 1.5 μg of protein. The bands at ≈340 kDa (*) and ≈260 kDa (¤) were excised and subjected to SDS-PAGE. Lane 4, sample from incubation of 5LO and ND without Ca2+. (B) SDS PAGE (12% Tris-glycine gel) denaturing analysis of the two bands excised from BN-PAGE lane 3. Both excised bands contain MSP1E3D1 (32 kDa) but only the ≈340 kDa (*) band contain 5LO (78 kDa).