20. Aluminum foil21. Agarose gel el

20. Aluminum foil21. Agarose gel electrophoresis system with incorporated power supply22. Agarose gel containing DNA-binding stain (already placed in electrophoresis system)23. Disposable gloves24. Goggles25. Water proof pen marker26. Three flags, colored red, green, and yellow27. Flag stand (=small tube) located on the left wall of your desk28. Your name tag (placed on shelf)We suggest that you familiarize yourself with the experiment by reading the entire text belowbefore starting the experimentIntroductionIn this experiment, you will test the ability of a protein named Pep (that is positively charged under theexperimental conditions) to interact with DNA. You will be supplied with the DNA to be tested, but youmust purify the protein Pep from a crude bacterial lysate. The bacteria had previously been transformedwith a plasmid expression vector into which the Pep encoding gene with a histidine tag had been cloned.Purification will be done by affinity chromatography. The histidine tag has affinity for and binds to nickelwhich is attached to the resin in the columns. After binding, the protein can be detached from the resinby changes of buffers used in the chromatography protocol. Eluted fractions will be collected in severaltubes. You will determine the protein concentration in two of the fractions by the Bradford method. Thisis a colorimetric assay in which attachment of Comassie brilliant blue to protein results in increasedabsorbance at wavelength of 595 nm. By using a standard curve derived by assay of a bovine serumalbumin (BSA) protein solution of known concentration, the protein concentration of the fractions can bedetermined. Subsequently, you will test the ability of the Pep protein in one of the fractions to interactwith DNA by performing a gel retardation assay. In this assay, interaction of DNA with protein retardsthe migration of the DNA on agarose gels during electrophoresis.