Buffer Preparations
(notes 3/3/04 jwc)

The main task today was the preparations of three buffers for the protein purification step. The homogeneity buffer was:
1) 25 mM HEPES
2) 0.1mM EDTA
3) 0.5 M NaCl

As 200 ml was required, 1.192 grams of Hepes, a dilution of .5 M EDTA stock using 400 microliters, and 5.844 gram of NaCl were prepared in about 130 ml of double deionized water chilled at 4 degree Celsius. After the solid material complete dissolved the level was raised to 190 ml and balanced to a pH of 7.5. Then the level raised to the required 200 ml, filtered and degasses in a sonic treatment. Finally stored in the cold room at the 4 degrees Celsius.

The equilibration buffer (Buffer A) was also prepared:
1) 20 mM HEPES
2) 0.5 M NaCl
3) 5 mM Imidazole
4) 2 mM Beta-mercaptoethanol

As 200 ml was required, .953 grams Hepes, 5.844 grams NaCl, 0.068 grams Imidazole, and 28 microliters of Beta-mercaptoethanol were prepared in about 130 ml of double deionized water chilled at 4 degree Celsius. After the solid material complete dissolved the level was raised to 190 ml and balanced to a pH of 7.5. Then the level raised to the required 200 ml, filtered and degasses in a sonic treatment. Finally stored in the cold room at the 4 degrees Celsius.

The elution buffer (Buffer B) was also prepared:
5) 20 mM HEPES
6) 0.5 M NaCl
7) 0.8 M Imidazole
8) 2 mM Beta-mercaptoethanol

As 200 ml was required, .953 grams Hepes, 5.844 grams NaCl, 10.89 grams Imidazole, and 28 microliters of Beta-mercaptoethanol were prepared in about 130 ml of double deionized water chilled at 4 degree Celsius. After the solid material complete dissolved the level was raised to 190 ml and balanced to a pH of 7.5. Then the level raised to the required 200 ml, filtered and degasses in a sonic treatment. Finally stored in the cold room at the 4 degrees Celsius.

This elution buffer will be used to elute the protein from the Ni-affinity column using the gradient of the Imidazole concentration.