Using The Baculovirus Expression Vector System ( Bevs ) Essay

Using The Baculovirus Expression Vector System ( Bevs ) Essay

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In this experiment, Professor Levinger has prepared expression vectors using the Baculovirus Expression Vector System (BEVS). Using his baculoviruses, we will prepare native Drosophila and Human tRNase Z proteins produced in insect cells. Invitrogen’s pFastBacHT vector for cDNA cloning is used. It creates a tag on the N-terminal peptide terminal chain on the cDNA which results in 6 consecutive histone residues. The 6XHis tagged binds to ions of Nickel (Ni2+) and ions of Cobalt (Co2+); in this laboratory experiment, Nickel is used as the binding agent. Dr. Levinger would pre-infect a flask of Sf9 insect cells with various stocks of Baculoviruses expressing tRNase Z polypeptides. The various stocks include non-mutated wild type Drosophila melanogaster tRNase Z (FFZ), Drosophila tRNase Z Carboxy terminal domain (TCdom), Human tRNase Z long from (HuZL), Human tRNase Z long form splicing isoform 3 with a 40 residue deletion (HuZLIso3), and Human tRNase Z short form (HuZS). Dr. Levinger already harvested the insect cells, which leads the class to start with frozen insect cell pellets. The pellets were then thawed to initiate the cell lysis procedure. The lysate was separated by centrifugation, in which the supernatant is applied to the Qiagen Ni-NTA Nickel affinity spin column. The column was then washed with wash buffer which elutes the 6XHis tagged tRNase. The eluted compound is frozen and stored for the next lab session. Samples of the crude cleared lysates and column flow-through is collected to track the progress of the affinity purification.
During the subsequent lab session, protein samples are loaded and ran on a Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). After the gel is done, it was stained with a f...


... middle of paper ...


...t viability can be calculated.1
Hypothesis-
Part I: Baculovirus/Insect Cell Expressed tRNase Z affinity purification-
During this procedure, I hypothesize that everyone in the class, for the most part, would efficiently elute the 6XHis Tagged proteins using the Qiagen spin column by using the proper buffers.
Part II: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis-
During this procedure, I hypothesize that if the preceding steps were done correctly the SDS gel would be able to show if the affinity purification worked properly and also show the relative size of the proteins.
Part III: Measuring Viable Cell Density Using a Hemocytometer-
During this procedure, I hypothesize that the cells that are dying would turn blue in color and cells that are healthy would be transparent. Thus, the cell viability could be calculated as well as the percent viability.

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