Glutathione's Transferase Lab Report

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Glutathione-S-Transferase (GST) is a detoxification enzyme which transfer glutathione to toxic substance and can be used as a tag when fused to a protein of interest. The purpose of this experiment was for us to determine which culture (A and B) was treated with Isopropyl B-D-thiogalactoside (IPTG) and therefore would express GST. The bacteria E. coli BL21 carrying the pGEX-2T plasmid was used to express GST protein after which protein extraction and GST purification from other proteins where done. GST expression is controlled by the Lac operon promoter (Plac).
The Lac promoter sequence drives GST gene expression (transcription) and the lac repressor gene encodes a protein that represses the lac promoter. When the lac repressor gene is always
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On our SDS-PAGE, our protein standards were separated alongside our protein samples which allowed determination of the sizes of our protein in our sample. SDS denature proteins and coats them with a negative charge allowing for their electrophoretic separation by polyacrylamide gel electrophoresis (PAGE). As you can see, after being stained with the coomassie blue, we could detect proteins of various sizes and our unknown protein expressing GST was detected in our Pure B sample (Figure 2). The distance travelled from the wells on the SDS-PAGES by our protein standards were measured and compared to our protein ladder in Daltons (Table 2), from which we could construct our semi log plot. On the semi-log plot, our unknown protein within our Pure B travelled 27mm from the well with a size of 29,000 Dalton (Figure 3). To identify the specific protein in the gel, the western blotting method was used to detect which of the cultures (A or B) contains GST (Figure 4). After the cassette was taken out of the electrophoresis unit, colors of our protein standards appeared on top of our membrane surface. After blocking, primary antibody incubation, and secondary antibody incubation, we could clearly identify our GST in our Pure B and Crude B (Figure
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