Buffers for protein purification:
1.Lysis buffer from yeast_cell_disruption.txt
2.Wash buffer : 50 mM NaH2PO4, 300 mM NaCl, 20 mM Histidine pH8.0 
3.Elution buffers:
a.50 mM NaH2PO4, 300 mM NaCl, 50 mM Histidine pH=8.0
b.50 mM NaH2PO4, 300 mM NaCl, 100 mM Histidine pH=8.0
c.50 mM NaH2PO4, 300 mM NaCl, 150 mM Histidine pH=8.0
d.50 mM NaH2PO4, 300 mM NaCl, 200 mM Histidine pH=8.0
e.50 mM NaH2PO4, 300 mM NaCl, 250 mM Histidine pH=8.0

Purification protocol:
1.The yeast lysate (end point solution from step II) was centrifuged at 10,000 g for 25 min at 4 C and the supernatant retained.
 (20 µl of the cleared lysate was retained for SDS-PAGE analysis- L.)

2.The Ni-NTA spin column was equilibrated with 600 µl lysis buffer and then centrifuged for 2 min at 700 g (approx. 2000 rpm). The spin column should be centrifuged with an open lid to ensure that the centrifugation step is completed after 2 min.

3.600 µl of the cleared lysate containing 6 x His-tagged protein was loaded onto the pre-equilibrated Ni-NTA spin column and centrifuged for 2 min at 700 g (approx. 2000 rpm) and the flow-through collected.
(20 µl of the flow-through was retained for SDS-PAGE analysis- FT.)

4.The Ni-NTA spin column was washed twice with 600 µl wash buffer and centrifuged for 2 min at 700 g (approx. 2000 rpm). 
(20 µl of each wash fraction was retained for SDS-PAGE analysis.-W1 and W 2)

5.The protein was euted in 5 steps of 200 µl of each elution buffer (a-e above). The column was centrifuged for 2 minutes at 700 g (approx. 2000 rpm) and the eluate collected after each step.
(20 µl of every eluate step was put aside for SDS-PAGE analysis-E1 to E5)

All eluates were stored in the fridge until the fraction(s) containing the recombinant His-tagged protein had been identified by SDS-PAGE analysis of all saved 20-µl samples.  Histidine was removed from the fractions thus identified by dialysis.