User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2012/02/22

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Objective

Continue preparing for the MBP-intein expression. Continue the concentration of the purified GFP. Reproduce results from 12/07/11 with gold nanoparticles and refolding buffer.

Description

Prep for MBP-intein expression

  1. Dry cycle in autoclave of various sizes of centrifuge tubes for expression next week.
  2. Combine 25g LB with 2g of D-glucose and 1L of distilled H2O in a 2800mL flask. (prepare 2)
  3. Combine 0.875g LB with 0.07g D-glucose and 35mL of distilled H2O in a 250mL flask. (prepare 2)
  4. Autoclave all of this media in a liquid cycle.

Studying the Effect of Refolding Buffer (repeat procedure from 12/07/11)

  1. Mix 100μL of 2.76mM HAuCl4 and 100μL 15.06μM BSA with 800μL distilled water in a microcentrifuge tube.
  2. Place the tube on a heat block at 80°C for three hours.
  3. Add 500μL 50mM Tris, pH 7.5.
  4. Remove from heat after about 5 min. Observe.

Continuation of Concentration

  1. Add more binding buffer (20mM sodium phosphate, 0.5M NaCl, 20mM imidazole) to concentrator, and continue concentrating the purified GFP to about 10mL.
  2. Sterile filter the concentrated GFP and store at 4°C.


Notes

  • Purple protein fibers formed before the solution was even removed from the heat block.



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