Team:Hong Kong HKU/BMCpurification

BMC Purification

BL21(DE3) cells carrying pETE and pET28a separately were inoculated and induced overnight using 0.2mM IPTG as described in the previous section, but in a liter scale. Sonication and clarification as performed as described previously, with all procedures post-sonication carried out at 4°C, and all solutions in contact with the sample and column was pre-chilled to 4°C. An adjustment solution* was added slowly with shaking to convert the composition of the sonication buffer into that of Ni-Binding buffer. The clarified, adjusted samples were filtered through a 0.45μm-pore membrane, and subsequently loaded separately onto 1ml HiTrap Chelating columns (GE Health Sciences). The column, bathed in 20% ethanol, was prepared by (1) washing with 5-columns of MiliQ water, (2) washing with 5-columns of Ni-Binding buffer, (3) loaded with 100mM NiSO₄ in Ni-Binding buffer, and finally (4) equilibrated with Ni-Binding buffer just before sample loading. After sample loading, the column was washed with 5-columns of Ni-Binding buffer, and elution was carried out in gradients of 30%, 50% and 100% Ni-Elution buffer* in Ni-Binding buffer, and finally stripped with Strip buffer*. To preserve the column for future use, the column was stripped with 5-columns of Strip buffer noting that the column of the column bed should be pure white, and washed with 5-columns of MiliQ water and re-bathed in 20% ethanol. All the elution and stripping fractions were collected, and protein concentrations were adjusted for normalization before loading on an SDS-PAGE for analysis, as described previously.

*2X Adjustment solution composition: 1M NaCl, 40mM Imidazole
Ni-Binding buffer composition: 25mM Tris-HCl, 500mM NaCl, 20mM Imidazole, pH 8.0
Ni-Elution buffer composition: 25mM Tris-HCl, 500mM NaCl, 500mM Imidazole, pH 8.0
Strip buffer composition: 25mM Tris-HCl, 500mM NaCl, 50mM EDTA, pH 7.4