Explain the principles behind each step in the following protocol:
1- Homogenize of the animal brain in the extraction buffer “buffer A” (0.1 M KH2PO4, 2 mM EDTA, 2 mM EGTA, 2 mM Dithiothreitol DTT, and 0.3 mM phenylmethylsulfonyl fluoride) pH6.9.
2- Centrifuge of the homogenate for 30 min at 13,000 X g. The resulting extract was brought to 45% saturation with ammonium sulfate and the pH adjusted to 7.5.
3- After 30 min, the mixture was centrifuged for 30 min at 13,000 X g. The resulting pellet was resuspended in a minimal volume of buffer A, pH 6.9, using a Dounce homogenizer and extensively dialyzed against buffer A, pH6.5, to remove residual ammonium sulfate.
4- The dialysate was then run on fplc using SP Sephadex C-50 previously equilibrated with buffer A, pH 6.5.
5- Wash the column with buffer A, pH 6.5, and the tau protein was eluted with buffer B (0.1 M KH2PO4, 2 mM EDTA, 2 mM EGTA, 2 mM DTT, and 0.3 mM phenylmethylsulfonyl fluoride, 0.5 M NaCl.) pH 6.5.