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

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Batch Purification, continued and SDS PAGE's

We continue troubleshooting our batch purification protocol, this time trying it out on another sample of lysate. We did notice that yesterday, when we first diluted our lysate 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. Decant supernatant.

4. Add 10 mL (about 2.5 BV) of 1X Binding buffer.

5. Centrifuge for 2 minutes at 700g at 4 degrees Celsius. Decant supernatant.

Batch Purification Protocol

1. Dilute 10mL of lysate sample in 10 mL of 1X Binding buffer.

2. Add to prepared resin. Spin on rotary spinner for 30 minutes at room temperature.

3. Centrifuge for 2 minutes at 700g at 4 degrees Celsius. Collect supernatant as Fraction 1.

4. Add 10mL of Wash buffer (20 mM imidazole) to resin. Spin on rotary spinner for 5 minutes at room temperature or until fully incorporated into resin.

5. Centrifuge for 2 minutes at 700g at 4 degrees Celsius. Collect supernatant as Fraction 2.

6. Add 10mL of Wash buffer (50 mM imidazole) to resin. Spin on rotary spinner for 5 minutes at room temperature.

7. Centrifuge for 2 minutes at 700g at 4 degrees Celsius. Collect supernatant as Fraction 3.

8. Add 10mL of Wash buffer (100 mM imidazole) to resin. Spin on rotary spinner for 5 minutes at room temperature.

9. Centrifuge for 2 minutes at 700g at 4 degrees Celsius. Collect supernatant as Fraction 4.

10. Add 10mL of Elution buffer (250 mM imidazole) to resin. Spin on rotary spinner for 10 minutes at room temperature.

11. Run resin contents through a gravity filtration column. Collect eluate as Fraction 5.

12. Add about 6mL (to the top of column) of Strip Buffer. Collect eluate as Strip Fraction.

13. To save resin for future use, add 10 BV of DI water, discard eluate. Then add 5 BV of charge buffer, discard eluate. Add 10 BV of DI water, discard eluate. Finally, add 5 BV of binding buffer, and drain into waste container until about 3mL remains above resin bed. Store at 4 degrees Celsius. For use next time, drain out buffer until only a little remains above resin bed surface.

SDS PAGE