Team:UCLA/Notebook/Recombinant Expression/27 July 2015

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Fraction 1 Re-purification using Standard Column Chromatography

1. Took 2mL from Fraction 1 of our batch purification conducted on Friday, and standard column chromatography is being conducted on the fraction to re-isolate Tamura protein for maximal yield.

Lyse the protein pellets from Friday's expression of MaSP2 9-mer using standard Alkaline Lysis

Each fraction was removed from -80 and treated with the following conditions:

1. Cells were resuspended in 100 uL cold GTE Buffer. 2. RNAse was added to a final concentration of 20 ug/mL 3. 100 uL of alkaline lysis buffer (NaOH, SDS) was added. 4. Incubate for 5 minutes on ice. 5. Add 100 uL of cold potassium buffer. Incubate for 5 minutes. 6. Centrifuge at 14,000g for 15 minutes at RT to pellet cell debris. 7. Mix 40uL of each fraction with 40uL of 2x Laemmli Sample Buffer 8. Heat treat samples for 5 minutes at 95 degrees. 9. Run SDS using 30 uL LSB at negative control, and 30 uL BSA as positive control (10 uL Dual Color Standards as Ladder)

304 uL of 2x LSB and 16 uL of Beta Mercaptoethanol to make final 2x LSB.

Pick colonies from the T7-sfGFP plate for miniprep.

To do tomorrow:

1. Make more imidazole stock solutions. 2. Do a Pierce Conc + BCA + Run 3 gels with all the fractions (9-mer, 1st purification, 2nd re-purifcation using column). 3. Pick up reagents and conduct the lysis of the 9-mer. 4. Miniprep T7-sfGFP plate colonies.