Proteome fractionation and purification flow chart.

<p>Approximately 500 g of <i>E. coli</i> cells were lysed at pH 7 using a microfluidizer and the cell debris pelleted. The supernatant was applied to a tangential flow column with a nominal molecular weight cut off of 500 kDa, generating 2 fractions (retentate and flow through). The fraction above 500 kDa (retentate) was further purified via sucrose gradients, size exclusion, and ion exchange chromatography prior to crystallization trials. The fraction less than 500 kDa was applied to multiple affinity and ion exchange columns followed by phenyl sepharose, ion exchange, and size exclusion prior to crystallization trials in microfluidic chips.