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Green Fluorescence Protein Lab Report

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The Expression and Purification of Recombinant Green Fluorescence Protein (rGFP) from E. coli using Ni+2 -Agarose Affinity Chromatography Abstract The purpose of this series of experiment was to express and purify His6tagged recombinant Green Fluorescent Protein (rGFP) in E. coli strain using Ni+2Agarose Affinity chromatography column. The strain BL21(DE3) < pRSETA-GFPUV> was induced and expressed as an N-terminal His6/Xpress epitope tagged fusion protein, the rGFP crude extract was then purified through Ni+2 agarose affinity column, with wash and elution fraction collected to analyze the fluorescent activity of rGFP under UV light. Green fluorescence observed indicated that rGFP was expressed in E. coli strain. Elution fraction E3 showed …show more content…

Bradford assay data indicated that the highest fluorescence activity in elution fractions was found in E3 fraction (2747.5 RFUs) and the highest protein amount was in E3 fraction with 29.61 ug and its specific activity was calculated to be 463791.4 RFUs/mg. Figure 4: SDS-PAGE Gel of rGFP samples (G0, G3, GCE, W2, W3, E2, E3, ladder) using 12% Resolving Gel and 5% Stacking Gel and stained with Coomassie Blue Figure 4 showed the SDS-PAGE gel made with 12% resolving gel and 5% stacking gel. SDS-PAGE gel was used to analyze the purity of rGFP from a crude extract, and the relative molecular weight. Standard molecular weights of ladder were 97.4, 66.2, 45, 31, 21.5, and 14.4 kDa. The estimated molecular weight of purified rGFP was calculated to be 33.48kDa, relative molecular weight of rGFP was 36kDa. By comparing the band density, the purity of rGFP was estimated to be about 40% for E3. Based on the purity percentage and the total protein amount, the estimated total yield of rGFP obtained was calculated to be …show more content…

coli strain using Ni+2Agarose Affinity chromatography column. These serial experiments were accomplished and the results were in acceptable range. The highest relative fluorescence activity was in elution fraction E3 as expected. The highest total protein amounts were found in the wash fractions as expected because non-rGFP protein was washed out and the rGFP was expected to come out in the elution fractions. The highest percentage of purity obtained was about 40% in E3 fraction, which should probably be higher with more skillful performance in the progress. The estimated molecular weight of rGFP was calculated to be 33.48 kDa and was found to be 36 kDa using SDS-PAGE. The western blot also confirmed the presence of rGFP, though there was slightly likelihood of contamination or degradation because the molecular weight or rGFP was not totally closed to the expected value of relative molecular weight (~36 kDa). Overall, though the reported results were not as desirable as expected, the goal of these experiment was

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The diagram above represents what was discovered during the experiment. There are two spots, “Cinn.” meaning Cinnamaldehyde and Product referencing either (E,E)-1,4-diphenyl-1,3-butadiene or (E,Z)-1,4-diphenyl-1,3-butadiene. The blurring between the two spots for the crude product indicated that they were not pure and could have still had starting material leftover. There are two R_f values because from the UV analysis there was slight separation between the two spots, but they were still blurred together.

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