Team:UCLA/Notebook/Recombinant Expression/10 July 2015

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Batch Purification, continued and SDS PAGEs

We continue troubleshooting our batch purification protocol, this time trying it out on another sample of our lysate. We did notice that yesterday, when we first diluted our Lysis Buffer in 1X Binding buffer in a 1:1 volume ratio, coagulation and precipitation appeared. As the contents of the tube came closer to room temperature, coagulation and precipitation lessened, but still present. We suspect that the lysate sample used yesterday had excess contaminants that led to this unexpected precipitation. Today, when diluting our lysate sample in 1X Binding buffer, again in a 1:1 volume ratio, the solution became a bit cloudy, but no evidence of precipitation or coagulation. We interpreted this as a good sign and continued on with our purification protocol.

Modification to Protocol : Instead of centrifugation after adding the Elution Buffer (250 mM Imidazole), we used gravity filtration to filter out the solvated Tamura proteins from the resin pellets. After checking this filtrate under a UV box, it glowed green, verifying the presence of solvated Tamura.

Resin Preparation

1. Add 4 mL of Histidine resin to a 50mL Falcon tube.

2. Add 8 mL of DI water.

3. Centrifuge for 2 minuts at 700g at 4 degrees Celsius.

SDS PAGE