The sample after incubating was centrifuged at 3000 rpms for 7 minutes, room temperature. To isolate the protein, the supernatant was poured off and the pellets were resuspended in 5 ml of lysis buffer (8 M urea, .1 M NaH2PO4, .01 M Tris Cl, pH 8.) The sample was then incubated at room temperature for 40 minutes centrifuged at 10000rpms for 20 minutes to separate protein from other cell debris. 50% Ni-NTA (Nickle coated beads, solution) was added and shaken for 15 minutes at room temperature and spun down at 1000rpms for 5 minutes. The pallet was then washed with buffer C (8 M urea, .1M NaH2PO4, .01 tris-Cl, pH 6.3) and centrifuged for 1000rpm for 5 minutes. The beads were spun down at 1000 rpms for 5 minutes to remove as much of the supernatant as possible. Finally, the pallet was suspended in 500 microliters of buffer E (8 M urea, .1M NaH2PO4, .01 tris-Cl, pH 4.5.)
The samples above were run on an SDS gel for one hour at 100 volts, after which it was stained with coomassie blue (45% methanol, 10% acetic acid, .1g coomassie blue.) De-staining was accomplished in 45% methanol, 10% acetic acid.)
Between many of the purification steps, samples were saved to test for presence of protein. Pallets (insoluble bacteria membranes), lysate (contains the soluble bacteria proteins), flow through (the lysate after incubation of the nickel beads), wash (the buffer C used to rinse the beads bound with xb10, protein (xb10), beads (after elution with buffer E.) (Elution, protein come off beads and back into solution.)
