Continue sperm whale myoglobin expression as started by Tamra notebook page and finish purification of protein as started yesterday notebook page.
Start WT Hemoglobin Expression
- Inoculate starter cultures (35mL LB, autoclaved) with 35uL of 100mg/mL Ampicilin and cells from WT Ascaris Hemoglobin glycerol stock in -80 freezer.
- Shake/incubate the cultures overnight at 37C
Continuing Myoglobin Expression
- Spin down cells in starter culture (20 minutes, 4000 rpm, 4C)
- Prep expression culture media
- Add autoclaved phosphate to autoclaved TB with sterile technique.
- Add 1mL of 100mg/ml Ampicilin
- Innoculate expression cultures with resuspended cell pellets from starter cultures.
- Shake at 165rpm and 37C until OD reaches 6.6
Purification of WT Ascaris Hemoglobin
- Equilibrate Q column with water and then with Buffer A (25mM Tris, 50mM NaCl, pH 8)
- Put sample on superloop and inject sample onto column
- 200mL of sample. Adding to column will take longer than desired
- Run 25mL of Buffer A over column after injection finishes
- Start Eluting
- Buffer B (25mM Tris, 1M NaCl, pH 8)
- Gradient: 0-50% Buffer B in 30 minutes
- Collect Fractions
- Add data and results here...
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