Method for Recovery and Immunoaffinity Enrichment of Membrane Proteins Illustrated with Metastatic Ovarian Cancer Tissues

Figure 2

A series of western blots for various membrane, nuclear, and cytosolic proteins (Table 2) extracted from metastatic ovarian tumor samples. The extracts were prepared using 3 different extraction buffers (lane B = HEPES buffer extract, lane D = TD1 buffer extract, and lane F = TD2 buffer extract). Lane H is a duplicate of lane F at 1/3 the protein loading. The corresponding insoluble proteins trapped in the pellets recovered after each extraction are also shown in the adjacent lanes (lane C = HEPES pellet, lane E = TD1 pellet, and lane G = TD2 pellet). Either a purified recombinant protein control (lane I) or a human cell line extract control (lane J) was used in each blot (both were used for EDG4, NRP1, and KDR). Lanes A (see blue, Invitrogen) and K (Magic Mark, Invitrogen) are molecular weight markers. Lane A is only visualized in visible light, not in the chemiluminescent images shown. Both of these markers (see blue from a white light image not shown) were used to determine the molecular weights shown (on the right hand side). Gaps between lanes are provided only for alignment purposes, and the lanes are from the same gels.