Figure 4

Immunoblot analysis of samples fractionated by density gradient centrifugation followed by two phase affinity partitioning using 6.3%PEG/Dextran. A - Fractionation of microsomes prepared from heads. Most Nervana is left behind in the dextran fractions, although a 60× exposure shows that a low yield of Nervana is found in the eluted ConA fraction (arrowhead in inset). BiP is no longer detectable in this combined preparation, demonstrating the utility of pre-fractionation on an Optiprep gradient. Residual ATP synthase is detected in the eluted fraction. Labels: Pool - pooled fractions 15-20 from the initial Optiprep gradient. Other labels are as described in figure 2A. Loading: Equivalent amounts of all fractions were loaded. Detection: normal sensitivity chemiluminescence (Nrv), high sensitivity chemiluminescence (BiP, ATP synthase). B - Fractionation of microsomes prepared from 0-15 hr embryos. Some α-Spectrin is recovered in the eluted fraction although some seems to have partitioned into dextran phase D1 and some has been excluded from the ConA as seen in the PEG phase (P1+P2). Neither BiP, nor ATP synthase is detectable in the eluted ConA fraction. Labels: same as A. Loading: Equivalent amounts of all fractions were loaded. Detection: high sensitivity chemiluminescence (α-Spectrin, BiP and ATP synthase). N.B. 'high' sensitivity detection reagents are 100-1000× more sensitive than the 'normal' sensitivity substrates and were used to detect even low-level residual contamination.