Figure 5
The dimeric form of immature α S1 -casein binds to ER membranes more efficiently. A. Aliquots of the microsomes were diluted and incubated in non-conservative buffer (NC) or were subjected to carbonate extraction at pH 11 (pH 11), both with saponin, in the absence (-DTT) or the presence of 5 mM DTT (+DTT) and centrifuged. Supernatants (S) and pellets (P) were analyzed by SDS-PAGE followed by immunoblotting with anti-mouse milk proteins. The quantity of immature αS1-casein in the supernatant and the pellet was quantified by densitometry and expressed as percent of the total quantity of the casein (supernatant + pellet). The mean ± s.d. of three independent experiments is shown. B. Microsomes were treated as above, all in the presence of DTT, and extracted membranes were floated using linear sucrose gradient. Fractions were collected from the top (light sucrose) to bottom (heavy sucrose) and analyzed by SDS-PAGE followed by immunoblotting for the indicated markers. Representative gradients from two independent experiments are shown. Relative molecular masses (kDa) are indicated. Cnx: calnexin, im. αS1-cas: immature αS1-casein.