Green Fluorescent Protein Lab Report

Expressing and Purifying the Recombinant form of Green Fluorescent Protein (rGFP) from the E.coli strain using Ni2+ agarose affinity chromatography technology
Abstract
The purpose of this experiment was to express and purify the his6-tagged recombinant form of GFP (rGFP) from the organism E.coli using Ni2+ agarose affinity chromatography. The expression of rGFP was confirmed qualitatively using the UV light and was expressed in the E.coli strain BL21 (DE3) (-- removed HTML --) (-- removed HTML --) . Ni2+ agarose affinity chromatography was used to purify the crude extract and different elusions were collected using the Ni2+ agarose column and then were analyzed qualitatively using UV light and quantitatively using fluorescent microplate reader
A sample amount of solution protein was made with the respective amounts knowing the concentration of the BSA stock solution, and Bradford reagent was added to the samples, giving a final volume of 250ul. These solutions were inoculated, and in a microplate reader the absorbance was measured at 595nm. A standard curve was created by plotting the absorbance (595nm) vs BSA (ug) data, and a best fit line was drawn. Then, absorbance readings at 595nm were triplicated using W1-W6 and E1-E6 sample fractions. Extrapolated the absorbance values on the standard curve and determined the amount of total protein that was present in the known volume of sample. (Source: See Lab
Added Ponceau S stain and on a shaking platform, incubated the nitrocellulose. Discarded the stain and rinsed the nitrocellulose several times with ddH2O until red protein bands appeared. Marked the MW ladder with a pencil. Placed two membranes facing outward in a blue Tupperware container. Added blocking solution (5% non-fat dry milk/TBS/0.05% Tween-20) and incubated the membranes in a rocking motion. Discarded the blocking solution and added mouse anti-Xpress epitope Mab solution to the membrane and incubated on a shaking platform. Then, anti-Xpress epitope solution was discarded and TBS solution was added to the membrane and incubated on a shaking platform. Discarded the wash solution and repeated this wash process two more times. Added Sheep anti-mouse IgG conjugated horse radish peroxide polyclonal anti-serum solution and incubated for on a shaking platform. Discarded the anti-mouse IgG solution and added TBS and incubated on a shaking platform. Discarded the wash solution and repeated thhis process. Finally, used a white Tupperware container filled with tap water and added TMB substrate solution to the membrane and incubated on a rocking platform until bands of rGFP became visible (Source: See Lab