Separation of erytrocyte memrane proteins with ammonium sulfate

In the present study, erythrocyte membrane proteins were precipitated by ammonium sulfate and then their electrophoretic mobilities were analysed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) using Laemmli slab gel discontinuous buffer system. For this reason, different methods were examined. 30, 10 and 10 normal samples were used for the first, second and third methods respectively. In the first method, ammonium sulfate saturation of 10%, 20%, 30%, 40% and 50%, and gel with 10% acrylamide concentration were used. The saturation of 10% was the best in which 95.5%, of the all bands were observed. For the second method, 5%, 10%, 15%, 20%, 35%, 30%, 40%, 50%, 60%, 70% and 80% saturation, and for the third method, 5%, 15%, 25%, 35%, 45%, 55%, 65%, 75% and 85% saturation were used to obtain erythrocyte membrane polypeptides. Both of the later methods were analysed by gels with 8% acrylamid concentration and 15% saturations were the best for both. However, abundances of then- protein bands were different such that while 88.2% of all bands were observed with 15% saturation in second method, 92.7% of all bands were observed with again 15% saturation but in third method. In all of three methods, it was observed spectrin dimer in almost all saturations. Moreover, the subunits of ankirin, protein 4.1, 4.2, and 4.5 could be seen. Protein 4.2 was seen as dimer especially with 10% saturation in the first method. In addition, protein 4.5 was sometimes seen as trimer. To determine the effect of EDTA on protein separation, reservoir buffers with EDTA and without EDTA were used. In the result of electrophoresis, reservoir buffer with EDTA was better than the other due to the observations of bands.