Lab 2 Protein Purification

Lab 2 Protein Purification - Zachary Bergman Dallas 2/12/09...

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Zachary Bergman Dallas 2/12/09 Protein Purification Introduction The goal of this lab was to purify and segregate the protein ICER from a mixture of proteins using a variety of techniques. The effectiveness of the different techniques were analyzed using multiple tests which were able to compare the isolation of the ICER. The tests that were used were SDS-PAGE, western analysis and an electrophoretic mobility shift assay. Each was able to presence of the ICER protein as well as the purity of the sample. Multiple tests were used both to confirm the accuracy of one another as well as to teach the possible ways of determining protein presence and purity. Materials and Methods Part IA: Protein Purification: The bacteria expressing the ICER was initially sonicated to lyse the cell walls and free the protein. The ICER was then purified by repeatedly using a centrifuge. Three fractions of the protein were taken during the purification process which are tested in the later parts of the experiment. Fraction 1 was simply the original ICER after a single centrifuge which removed the bacteria cell walls. Fraction 2 was obtained after an addition of His/Bind Resin and mixing for 30 minutes. Fraction 3 was the final purification which took Fraction 2 and added a series of buffers, including a Binding Buffer and Elution Buffer, then mixed again for 30 minutes. After both Fraction 2 and 3 the solution was centrifuged to remove any solids. Part IB: Polyacrylamide Gel Electrophoresis: The acrylamide gel was made and placed between the plates once they were even and properly secured. The comb was then inserted into the gel in order to make the individual lanes which the samples are loaded into. Once the gel was dry, student samples of Fractions 1, 2, and 3 as well as a control were loaded into separate lanes. The electrophoresis was then run at ~200 volts and then stained, a picture of the developed gel was then taken. Part IIA: Electrophoretic Mobility Shift Assay:
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Lab 2 Protein Purification - Zachary Bergman Dallas 2/12/09...

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