Lux G Experiment
Lux G experiment is performed at the University of California San Diego on May 21st until June 4th of 2018 by Gloria Karem. This experiment is based on the purification of LuxG by using multiple purification and lysing methods: Brad Ford Assay, SDS-PAGE, affinity purification, lysozyme treatment, nickel column, sonication, and flavin reeducate activity assay. His-tagged protein is expressed in E. coli from pGhis, with the T7 RNA lac repressor induction system.5 Sonication is used, it’s why bacteria are lysed. The tag allows purification for ion affinity chromatography, following a gel filtration column, defined as removing ions and other small molecules. Where then, the Bradford assay was made use of to identify the protein concentration from
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coli resulted to two total amounts of proteins: the heart crude and the purified extract. The heard crude had a total amount of 303.77g, and the concentrated pooled protein had a total amount of 86.5 mg/ml. The concentration was calculated for the heart crude when using 30uL to be 181.3 mg/ml, and for the 60uL used having a concentration result of 480.10 mg/ml. The volume(mL) of the crude extract is found to be 10.2mL, the mass is found to be 303.77g, the protein concentration is found to be 29.78mg/ml, the total activity is calculated to be 5.9µmol/min, the activity concentration is calculated to be 0.58 µMol/min*mL, the specific activity(µmol/min*mg) is calculated to be 0.019, and the turnover number is calculated to be 0.009, 0.02mL of enzyme was added to the crude extract with a total volume of 0.25mL. The purified extract is found to be 0.30mL, the mass is found to be 8.23g, the protein concentration is found to be 35.31mg/ml, the total activity is calculated to be 0.65µmol/min, the activity concentration is calculated to be 2.6µmol/min, the specific activity(µmol/min*mg) is calculated to be 0.07, and the turnover number is calculated to be 0.03, 0.09 mL of enzyme is added to the purified extract with a total volume of purified protein of 10.2mL. The molecular weight of the LuxG is calculated to be 28360 g/mol. The molecular weight of LuxG-his6 from SDS-PAGE is calculated to be 34.6
coli resulted to two total amounts of proteins: the heart crude and the purified extract. The heard crude had a total amount of 303.77g, and the concentrated pooled protein had a total amount of 86.5 mg/ml. The concentration was calculated for the heart crude when using 30uL to be 181.3 mg/ml, and for the 60uL used having a concentration result of 480.10 mg/ml. The volume(mL) of the crude extract is found to be 10.2mL, the mass is found to be 303.77g, the protein concentration is found to be 29.78mg/ml, the total activity is calculated to be 5.9µmol/min, the activity concentration is calculated to be 0.58 µMol/min*mL, the specific activity(µmol/min*mg) is calculated to be 0.019, and the turnover number is calculated to be 0.009, 0.02mL of enzyme was added to the crude extract with a total volume of 0.25mL. The purified extract is found to be 0.30mL, the mass is found to be 8.23g, the protein concentration is found to be 35.31mg/ml, the total activity is calculated to be 0.65µmol/min, the activity concentration is calculated to be 2.6µmol/min, the specific activity(µmol/min*mg) is calculated to be 0.07, and the turnover number is calculated to be 0.03, 0.09 mL of enzyme is added to the purified extract with a total volume of purified protein of 10.2mL. The molecular weight of the LuxG is calculated to be 28360 g/mol. The molecular weight of LuxG-his6 from SDS-PAGE is calculated to be 34.6