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Purification of HQ-Tagged Proteins 155
detergents (lysis buffers). We have described lysis methods using sonication
and lysis reagents (see Notes 1 and 2).
3.1.3. Purification
HQ-tagged proteins can be purified using different resins and formats
from small to large-scale, manual or high-throughput and magnetic and nonmagnetic.
3.2. Lysis and Purification from Bacterial Culture Using
Magnetic Ni Particles
Lysis of bacterial culture can be done in culture or using pelleted cells;
however, the purification protocol is the same for either lysis method.
3.2.1. Lysis of Pelleted Bacterial Cells Using Lysis Buffer
1. Centrifuge 1 ml of bacterial culture at 14,000 rpm for 2 min in a microcentrifuge.
Remove the supernatant completely.
2. For every 1 OD 600 nm of culture, dilute 10 μl FastBreak Cell Lysis Reagent, 10×,
to 1× by adding 90 μl NANOpure® or double-distilled water (100 μl total).
3. Resuspend the cell pellet in 100 μl 1× FastBreak Cell Lysis Reagent for every 1
OD 600 nm (for example, for a3OD 600 nm culture, use 300 μl 1× FastBreak Cell
Lysis Reagent).
4. Resuspend lyophilized DNase I as indicated on the vial (see Note 3) and add 1 μl
to the bacterial culture.
5. Incubate with shaking for 10–20 min at room temperature on a rotary mixer or
shaking platform to lyse bacteria.
3.2.2. Direct Lysis of Bacterial Cell Cultures Using Lysis Buffer
1. Add 110 μl of FastBreak Cell Lysis Reagent, 10×, (1/10 volume) directly to 1
ml of fresh bacterial culture, OD 600 nm