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IreneKatseftisLR2 - Irene Katseftis Biology 3096 Lab Dr...

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Irene Katseftis Biology 3096 Lab Dr. Angela Bricker October 19, 2011 Objective: In order to detect proteins in the fractions, four substances were separated using gel filtration. Using gel electrophoresis by SDS P.A.G.E, students analyzed their protein fractions, and estimated the molecular weights of their proteins. Methods 1 : In the first part of the experiment, students performed gel filtration, which is a method of column chromatography that separates molecules by size. Working in groups of two, students poured the column by adding liquid from above the gel slurry (slurry of Bio-gel P-60 beads in water) to the column. The slurry was added until the column reached the desired height of 10 to 12 cm. Once the liquid level dropped to the top of the column, the sample [mix of 2 mg/ml Blue Dextran (blue, 2 MDa), 5 mg/ml potassium chromate (yellow, 194 Da), 2 mg/ml BSA in phosphate buffer] was then added to the top of column and the protein fractions were collected. Blue Dextran is a very large polymer of anhydroglucose coupled to Reactive Blue 2 dye, which was necessary for determining the column’s void volume. The potassium chromate was a small molecule that enabled students to determine the column’s entire volume. As the 0.5 ml fractions were being collected, students added equilibration buffer (20 mM phosphate buffer pH7 – 7.4) to the column as the liquid level fell. While one student obtained the fraction samples, the other student spotted 1 l of each fraction onto nitrocellulose filter. Once all the fraction samples were spotted and the filter was dry, the 1
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filter was incubated in 50% methanol for 5 minutes. The filter was then incubated again in amido black (0.1% amido black 10B in 30% methanol-10% acetic acid) for 5 minutes. Lastly, the filter was rinsed twice with 50% methanol until the background no longer changed. The amount of dye that bound to the filter was proportional to the amount of protein present. The students assayed the protein content of their gel filtration fractions obtained. In the polyacrylamide gel used, the stacker was more porous that the resolving gel (4% T in stacker, 12% T in resolving gel). The fractions used were the ones that Blue Dextran started to emerge until the time that the potassium chromate started to emerge. The student mixed 20l of each fraction with 20l 2X Sample buffer (containing and SDS) and heated them at 95 o C for 2 minutes in order to break the disulfide bonds. Before the
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