Green Fluorescent Protein Lab Report

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Expression and Purification of His6-tagged recombinant Green Fluorescent Protein (rGFP) from E.coli Strain using Ni+2 - Agarose Affinity Chromatography

Abstract
Green fluorescent protein(GFP) is a unique bioluminescent protein found in the light organs of Aequorea victoria, commonly known as crystal jellyfish, and which will have broad uses in cell and molecular biology as a reporter of gene expression. The purpose of this experiment was to express a recombinant form of GFP in the E.coli strain BL21(DE3) and purify it using Ni+2 agarose affinity chromatography technology. The GFPUV gene containing an N-terminal, Xpress epitope tag was induced and overexpressed in E.coli, making a crude extract. This was then purified using Ni2+-agarose affinity …show more content…
The curve would be later used as a reference to interpolate the amount of total protein in needed elutions. Knowing the concentration of BSA stock solution to be 0.5mg/ml, the volume of each different BSA assay was calculated. Water was added to each microplate containing an assay until a volume of 50ul was reached and then 200ul of Bradford dye was also added. The solutions were incubated at room temperature for 10 minutes and their absorbance at 595nm was then measured. The six individual absorbance values were plotted against their mass to be used to create a standard curve with a line of best fit, and act as reference for the unknown amount of total protein in the twelve fractions. Similarly, Bradford microplate assays were performed in triplicate for the twelve fractions and the absorbances were interpolated on the standard curve to determine the amount of total protein present in each sample …show more content…
20ml of Ponceau S stain were added and the nitrocellulose was incubated with a rocking motion for 2 minutes. The stain was then discarded while the paper was washed with ddH2O. The molecular weight ladder was marked and 30ml blocking solution (5% non-fat dry milk/TBS/0.05% Tween-20) was added and incubation occurred on a shaking platform for 30 minutes. Primary antibody composed of 7ml of mouse IgG anti-Xpress epitope MAb solution replaced the blocking solution while the membrane was shaken for 45 minutes. Afterwards, a wash with 30ml of 0.05% Tween 20/TBS replaced the primary antibody and the membrane was incubated on the shaking platform for 5 minutes. This step was repeated two more times. The secondary antibody composed of 7ml Sheep IgG anti-mouse IgG conjugated horse radish peroxidase polyclonal anti-serum was then added and the container shaken for 45 minutes. Another two washes followed the secondary antibody. Finally, 30ml of TBS were added as the membrane was incubated for 5 minutes. 7ml of TMB substrate solution were added to the container with the membrane and incubation with a rocking motion followed bands could be visualized. The membrane was then added in a container with tap water to stop the development process and then dried while the result was
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