Electrophoretic Analysis

If complex mixtures are analyzed, a lot of information can be obtained by a proper sample preparation and subsequent SDS-PAGE. In Figure 2, difference between calcium binding proteins in the plasma membranes of rat liver (left) and fast growing Morris hepatoma 7777 is shown. These proteins have been identified as annexins. Note the increased levels of annexins with lower molecular weights (between 30 and 35 kDa) in the extract from Morris hepatoma 7777 plasma membranes.

complex mixtures and subsequent SDS-PAGE

Figure 1

SDS-PAGE of rat liver plasma membranes. Membrane proteins were solubilized with one percent CHAPS in the absence of (left) or presence of EDTA (right). Arrows - proteins which are extracted after calcium complexing.

Left – Buffer with 2 mM CaCl 2.

Right – Buffer with 10 mM EDTA.

Figure 2

SDS-PAGE of calcium binding proteins (annexins) from plasma membranes of rat liver (left) and Morris hepatoma 7777 (right).

Other proteins have been removed by previous textraction with one percent Triton X100.

However, 2-D electrophoresis can yield much more information. As shown in Figure 3, the two lower bands from the SDS-PAGE in Figure 2, contain more than 10 polypeptides with very similar molecular weights that can be distinguished by differences in pI.

Figure 3

Two-dimensional electrophoresis of calcium binding proteins (annexins) from plasma membranes of Morris hepatoma 7777.

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