Fig. 2.

Comparison of the purity of membranous and cytosolic fractions by Western blotting of fractionated colon cancer cells using specific antibodies to each sub-cellular fraction. (A − C) membrane-specific and (D + E) cytosol specific antibodies. Cyt cytosolic fraction, Mem membranous fraction, vs versus. Kit I retains all three membrane proteins and shows a fairly good separation of those compared to the cytosolic fraction. However, the membranous fraction shows substantial contamination with both cytosolic proteins. Kit II only retains two of three membrane proteins and one of them is also detectable in the corresponding cytosolic fraction. In contrast, the cytosolic proteins are well separated. Kit III shows an almost perfect separation of both, the membranous and cytosolic fraction with minimal cross-contamination. Kit IV shows overall poor separation characteristics with strong cross-contamination of both sub-cellular fractions. Kit V poorly retains proteins of the membranous fraction. Both cytosolic proteins could be well separated with negligible cross-contamination of the membranous fraction. Reproducibility of replicates was between 73% (Kit IV), 80% (Kit I and II), 87% (Kit III) and 100% (KitV)