Analysis of soluble/insoluble partitioning of E. coli overex

Friday, November 21, 2003

Description
Analysis of soluble/insoluble partitioning of E. coli overexpressed proteins

Procedure
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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Analysis of soluble/insoluble partitioning of E. coli overex