Team:UCLA/Notebook/Recombinant Expression/23 July 2015

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IMAC Purification, Take Two

2mL of fresh resin was added to the a pre-chilled 50mL Falcon tube. The contents were centrifuged to discard the ethanol supernatant. The standard IMAC Batch Purification protocol was followed, as in previous purification trials.

Observations

The lysate seems quite viscous, and seems to have some stringy, thick and gluttonous strands suspended in the middle. Even when not observed under blue light, the lysate solution appears an obvious light green color. We hypothesize that this may be due to the super-folded GFP being super concentrated in the lysate. Also, when lysate, diluted in 1X binding buffer, is added to the resin and the first wash buffer is added (20mM imidazole), the resulting solutions appears white, and a bit foamy or soapy. This contrasts with our previous experience with IMAC Batch purification, when the solution appeared mostly clear. We also attribute this to our lysate being super concentrated, especially when compared to our previous batch of Tamura, which was suspended in excessive amounts of lysis buffer.