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 Experimental Biological Chemistry I
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Purpose
- To continue cell amplification from yesterday for greater yield of ADA proteins
- To continue test out the best concentration of reactants for luminol-peroxide reaction in addition to horseradish peroxidase
Procedure
- 0.4M IPTG for protein expression were made by mixing 0.4g of IPTG with 1mL of sterile water.
- In the morning, cells left in shaker were taken out and spin in centrifuge at 4500rpm for 15 minutes. Pellets form, and supernatant were discarded. Pellet were resuspended in 4mL of LB. 1mL of each cell culture were placed in 1L autoclaved sterile LB broth, 1mL of 0.4M IPTG were added before incubation. Cell culture were placed in shaker under 225rpm at 37C for ~4 hours.
- Elution and binding buffers were made. See Dhea's Notebook for the details in making elution and binding buffers used for future cell resuspension and protein purification.
- After 4 hours in the shaker, cells were transferred into centrifuge bottles with mass ~1 grams apart. Cells were centrifuged at 4500rpm for 15 minutes at 4C. Supernatant were discarded, pellet were resuspended with 30mL of binding buffer. Cell resuspension were stored in -80C until next week. This process is repeated two times for more cells.
- Luminol-peroxide reaction were further experimented. Luminol concentration were altered, and concentration of horseradish peroxidase were altered. For details of the experience, and details of data obtained, see Melissa's Notebook for information on concentration of luminol, H2O2, and HRP assay used in each trial. For initial and final concentration of the solutions, see below table:
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Initial Concentrations [mM]
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Final Concentrations [mM]
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| Trial |
4-Iodophenol |
Luminol |
H2O2 |
HRP |
4-Iodophenol |
Luminol |
H2O2 |
HRP
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| 1 |
18 |
1.25 |
1.7 |
0.0023 |
0.000119 |
0.000578 |
0.000846 |
7.63E-08
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| 2 |
18 |
0.625 |
1.7 |
0.0023 |
0.000119 |
0.000289 |
0.000846 |
7.63E-08
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| 3 |
18 |
0.625 |
0.85 |
0.0023 |
0.000119 |
0.000289 |
0.000423 |
7.63E-08
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| 4 |
18 |
1.25 |
0.425 |
0.0023 |
0.000119 |
0.000578 |
0.000212 |
7.63E-08
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| 5 |
18 |
1.25 |
1.7 |
0.0023 |
0.000119 |
0.000578 |
0.000846 |
7.63E-08
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| 6 |
18 |
1.25 |
1.7 |
0.00115 |
0.000119 |
0.000578 |
0.000846 |
3.82E-08
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Notes
- Cells were amplified by transferring from 4mL LB broth to 250mL LB broth, then to 1L LB broth. This sequence of steps created ideal environment for cells to divide at fastest rate.
- Cells were centrifuged in order to seperate the cells from LB broth before transferring into new broth. That way cells placed in new LB broth were the most concentrated.
- Binding and elution buffers will later be used in protein purification. The imidazol concentration in both buffers were directed towards the purification of adenosine deaminase protein.
- It was still not concluded the concentration of luminol and peroxide concentration through experimenting the concentration reactants and enzyme. However, it was concluded that the increase of enzyme concentration increases reaction speed. It was suggested turning the lights off in lab room while injecting horse radish peroxide with the fluorometer running in order for fluorometer to catch the quick luminescence reaction. For details of experimented concentration of reactants and enzymes, see Melissa's Notebook.
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Experimental Biological Chemistry I
