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Analysis of soluble/insoluble partitioning of E. coli overexpressed proteins
- Pellet 1.0 mL of induced cell culture in a microfuge tube. Remove supernant.
- Resuspend the cells in 0.5 mL of MQ H2O.
- Sonicate the cell suspension on ice: 15 sec total sonication time (0.5 sec on/0.5 sec off), microtip, power =4.5 on Misonix sonicator.
- Pellet the insoluble material (2 min, 14 krpm, microfuge).
- Carefully remove the supernatant to a fresh tube (be careful not to remove any of the pellet!). This is the soluble fraction.
- Next, resuspend the pellet in 0.5 mL H2O.
- Centrifuge as in step 4.
- Remove the supernant; resuspend the pellet in 0.5 mL H2O. This is the insoluble fraction.
- For analysis by SDS-PAGE, run 10 uL each of the soluble & insoluble fractions (mix with 10 uL 2X SDS-PAGE sample buffer).
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