Affinity Chromatography Lab Report

The Expression and Purification of Recombinant Green Fluorescent Protein (rGFP) From E. coli strain, BL21(DE3), Using Ni2+-Agarose Affinity Chromatography

Abstract: The purpose of these series of experiments was to express and purify recombinant Green Fluorescent Protein (rGFP) from the E. coli strain, BL21(DE3) by beginning with its purification via a Ni2+-agarose affinity chromatography column. The His6 tag of the rGFP bound to the Ni2+-agarose column and washes and elutions were obtained, with elution 3 containing the most amount of fluorescence at approximately 12,000 RFUs. From here, a Bradford assay was performed in order to determine how much protein was in each sample and an SDS-PAGE/Coomassie Blue analysis was done to determine the size and purity of rGFP in the elution 3 sample. The sample came out to be about 34 kDa and was about 75% pure. Lastly, a Western Blot was performed with binding of primary and secondary antibodies to prove that the protein of interest, rGFP, had indeed been expressed and purified.
Introduction:
Osama
Combined activity/elution Profile of rGFP during washes 1-6 and elutions 1-6 in a Ni2+-agarose column. The graph shows RFU’s (left) and total protein in ug (right) for each wash and elution. Once the rGFP had been purified in the Ni2+-agarose column, a breaking buffer (10mM Tris, pH 8.0; 150mM NaCl) was used to create the washes by pipetting the buffer in 0.5ml increments and collecting each in separate tubes labeling them W1-W6. Then, an elution buffer (10mM Tris, pH 8.0; 150mM NaCl; 300mM imidazole) was collected in increments of 0.5ml and labeled E1-E6. We could measure the amount of RFU’s each sample had using 200ul of each fraction and placing them in a spectrofluorometer. E3 showed to have the greatest amount of fluorescence. Using the Bradford assay, the total protein of each sample was determined by extrapolating points from the standard BSA curve. Both values were scaled