Difference between revisions of "Team:UCLA/Notebook/Recombinant Expression/9 July 2015"

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A new method of protein purification is discovered when searching online databases. Compared to gravity filtration through a nickel charged column, the advantages of this new method become apparent. Specifically, this method drastically decreases the amount of time needed to purify proteins, and allows for more homogenous mixing of sample with reagents added (binding, wash, and elution buffers). Batch purification shares the same general idea as column chromatography. Instead of a column, Histidine resin is placed in a Falcon tube and centrifugation is used to mix and separate contaminant proteins from our Tamura proteins. Instead of collecting what drips through the column as our fractions, after centrifugation, the supernatant is decanted from the tube and collected as fractions.

RESULTS

Our first attempt at batch purification appeared to be successful. After adding elution buffer (250 mM imidazole), we intended to take this supernatant (Fraction 5) and give it the members on the Materials Processing team to try to combine with recombinant silk to co-spin out silk fibers. However, Fraction 5 appeared to have a significant amount of precipitation and coagulation, mostly likely resulting from resin and excess protein residues. Fraction 5 also contained a great deal of thin, stringy white fibers, suggesting that our silk had "crashed out" and no longer solvated in Lysis buffer. Fraction 4, in comparison, was less cloudy, less precipitation and coagulation, and more transparent, indicating better solubility in buffer. Therefore, Fraction 4 was delivered to the members on the Materials Processing Team to do a preliminary co-spin with recombinant silk. Refer to Materials Processing, July 9 notebook entry to see images results! Overall, when checked under a UV box and a florescent microscope, our silk glowed green. The silk we extruded with the aid of a syringe pump is stretchy, but quite brittle. Optimization of protocols is the next step. We also need to create better controls to better justify our results. We did observe that our recombinant silk, when co-spun with purified Tamura protein, and observed under florescence microscopy, is thicker in diameter than our control.

Protocol

1. Chill a 50mL Falcon tube on ice. Add 2mL of resin slurry.

2. Centrifuge the tube for 2 minutes at 700g. Remove supernatant. NOTE: The speed was increased to 1,000g and then 3,000g to maximize the amount of ethanol supernatant.

3. Add 2 bed volumes (BV) of 1X binding buffer to resin and mix thoroughly on a rotary spinner until buffer is fully suspended in resin.

4. Centrifuge tube for 2 minutes at 700g. Remove buffer.

5. Add protein lysate diluted in 1X binding buffer in 1 to 1 volume ratio to tube. Mix for 30 minutes on an end over end rotation vehicle (rotary spinner).

6. Centrifuge the tube for 2 minutes at 700g. Decant supernatant and collect as Fraction 1.

7. Wash with 2 BV's of 1X wash buffer (20 mM Imidazole). Centrifuge for 2 minutes at 700g. Decant supernatant and collect as Fraction 2.

8. Wash with 2 BV's of 1X wash buffer (50 mM Imidazole). Centrifuge for 2 minutes at 700g. Decant supernatant and collect as Fraction 3.

9. Elute using 1 BV of elution buffer (100 mM Imidazole). Centrifuge for 2 minutes at 700g. Decant supernatant and collect as Fraction 4.

10. Repeat elution with 250 mM imidazole solution. Centrifuge for 2 minutes at 700g. Decant supernatant and collect as Fraction 5.